Antibodies against extracellular epitopes of the human TRPV6 channel and their diagnostic and therapeutic uses

Abstract

The present invention relates to an antibody against an extracellular epitope of human transient receptor potential vanilloid 6 (TRPV6) channel protein, in particular an antibody that regulates TRPV6 channel activity on the cell membrane, thereby inducing apoptosis of cancer cells expressing TRPV6. The present invention also relates to the use of this antibody for the diagnosis, prognosis, and treatment of diseases involving the TRPV6 channel, in particular diseases associated with TRPV6 expression, such as cancer. The present invention further relates to a peptide antigen derived from human TRPV6 protein that is useful for generating this antibody.

Description

[Technical field]

[0001] The present invention relates to an antibody against an extracellular epitope of human transient receptor potential vanilloid 6 (TRPV6) channel protein, in particular an antibody that regulates TRPV6 channel activity on the cell membrane, thereby inducing apoptosis of cancer cells expressing TRPV6. The present invention also relates to the use of this antibody for the diagnosis, prognosis, and treatment of diseases involving the TRPV6 channel, in particular diseases associated with TRPV6 expression, such as cancer. The present invention further relates to a peptide antigen derived from human TRPV6 protein that is useful for generating this antibody. [Background technology]

[0002] Transient receptor potential vanilloid subfamily member 6 (TRPV6) is a highly calcium-selective TRP channel that mediates calcium uptake in epithelial tissues and is involved in calcium homeostasis in the body (Clapham et al., Nat. Rev. Neurosci., 2000, 2, 387-96; Hoenderop et al., Pflugers Arch. 2003, 446, 304-8). TRPV6 is also known as transient receptor potential cation channel subfamily V member 6 (TrpV6), CaT-like (CaT-L); calcium transport protein 1 (CaT1) or epithelial calcium channel 2 (ECaC2). TRPV6 has four subunits that form a transmembrane domain (TMD) with a central ion channel pore flanked by intracellular N- and C-terminal domains. The TMD is composed of S1-S6 transmembrane helices and a concave pore loop (P-loop) between S5-S6 (Figure 1). TRPV6 is glycosylated at the N-glycosylation site located in the first extracellular loop (position 397 of the human TRPV6 sequence, FIG. 1). Human TRPV6 has the amino acid sequence of UniProtKB / Swiss-Prot NP_061116.5 or Q9H1D0.3 (SEQ ID NO: 1). The 3D crystal structure of TRPV6 has been determined (Saotome et al., Nature 2016, 534, 506-511).

[0003] Altered TRPV6 expression is associated with a variety of human diseases, including cancer. TRPV6 is highly expressed in endometrial cancer, leukemia, and carcinomas of the breast, pancreas, prostate, colon, ovary, and thyroid (Peng et al., Biochem. Biophys. Res. Commun., 2000, 278, 326-332; Wissenbach et al., J. Biol. Chem., 2001, 276, 19461-19468; Fleet et al., Am. J. Physiol. Gastrointest. Liver Physiol. 2002, 283, G618-G625; Zhuang et al., Lab. Invest., 2002, 82, 1755-1764; Fixemer et al., Oncogene, 2003, 22, 7858-7861; Wissenbach et al., Biochem. Biophys. Res. Commun., 2003, 277, 19461-19468). 2004, 322, 1359-1363; Taparia et al., Eur. J. Nutr. 2006, 45, 196-204; Wissenbach, U. & Niemeyer, BA, Handb. Exp. Pharmacol., 2007, 179, 221-234; Bolanz et al., Mol. Cancer Ther., 2008, 7, 271-279; Bolanz et al., Mol. Cancer Res., 2009, 7, 2000-2010; Semenova et al., Am. J. Physiol. Cell. Physiol., 2009, 296, C1098-C1104; Lehen'kyi et al., PLoS ONE, 2011, 6, e16856; Dhennin-Duthille et al., Cell Physiol Biochem., 2011, 28, 813-22; Zheng et al., Biochem. Pharmacol., 2012, 84, 391-401; Bowen et al., PLoS ONE, 2013, 8, e58866; Fecher-Trost et al., Handb. Exp. Pharmacol., 2014, 222, 359-384; Singh et al., Nature communications, 2018, DOI :10.1038; Song et al., Oncol Rep., 2018, 39, 1432-1440; Skrzypski et al., Biosci Rep., 2016, 36, e00372. doi: 10.1042; Masamune et al., Gastroenterology, 2020, 158, 1626-1641). Alterations in TRPV6 expression have been implicated in skin diseases such as psoriasis (Cubillos et al., J. Int J. Mol. Med., 2016, 38, 1083-92), alopecia and dermatitis (Bianco SD et al., J. Bone Miner. Res. 2007, 22, 274-85), epidermal proliferation disorders (Dai W et al., Cell Death Differ. 2014, 21, 568-81), skin aging (Li W et al., J. Gerontol. A Biol. Sci. Med. Sci., 2015, 70, 263-72), skin permeability barrier (Do BH et al., Acta Otolaryngol., 2017, 137, 1039-1045), hyperphosphatemia and ectopic calcification (Jurutka PW et al., J. Bone Miner. Res., 2007, 22 Suppl 2:V2-10), brain and nervous system disorders such as hearing loss and postpubertal goiter (Wangemann P et al., Am. J. Physiol. Renal Physiol., 2007, 292, F1345-53), preweaning (Lee GS et al., J. Bone Mine.r Res., 2007, 22, 1968-78), neuroexcitability (Brittain JM et al., Channels (Austin, Tex.), 2012 Mar-Apr;6(2):94-102), estrous cycle disorders and hypothalamic disorders (Kumar S et al., Neuroscience, 2017, 344, 204-216), circadian rhythm (Yang QJ et al., Drug Metab. Dispos., 2018, 46, 75-87), drug addiction (Janssens A et al., Pharmacol. Res., 2018, 136, 83-89), Parkinson's disease (Claro da Silva et al., J. Steroid Biochem. Mol. Biol., 2016, 163, 77-87), pain sensation (Jiang Y et al., Onco.l Lett., 2016, 12, 1164-1170), gastrointestinal disorders such as Crohn's disease (Huybers S et al., Inflamm. Bowel Dis., 2008, 14, 803-11), hypercalcemia (Zella LA et al., Endocrinology, 2009, 150, 3448-56), colonic crypt hypertrophy (Peleg S et al., Am. J. Physiol. Gastrointest. Liver Physiol., 2010, 299(3)), irritable bowel syndrome (Ishizawa M et al., Int. J. Mol. Sci., 2018, 19(7)), renal diseases such as arterial and renal calcification (Ignat M et al., Proc. Natl. Acad. Sci. US A., 2008, 105, 2598-603), chronic kidney disease (Torremade et al., PLoS One, 2017, 12, e0170654), bone mineral density and osteoporosis diseases and disorders (Bianco SD et al., J. Bone Miner. Res., 2007, 22, 274-85), gynecological disorders such as trophoblastic disorders (Bernucci L et al., Placenta, 2006, 27, 1082-95), female infertility (Yang H et al., Mol. Reprod. Dev., 2011, 78, 274-82), and diabetes mellitus (Lee CT et al., Kidney Int., 2006, 69, 1786-91).

[0004] TRPV6 has been implicated in tumor formation and progression, and this high expression pattern correlates with disease aggressiveness (Wissenbach et al., 2001; Fixemer et al., 2003; Wissenbach et al., 2004; Lehen'kyi et al., PLoS ONE, 2011; Peng et al., Biochem. Biophys. Res. Commun., 2001, 282, 729-734; Lehen'kyi et al., Oncogene, 2007, 26, 7380-7385; Lehen'kyi et al., J. Physiol., 2012, 590, 1369-1376).

[0005] Ca 2+TRPV6 is a key regulator of cell proliferation, suggesting a role for TRPV6 in enhancing calcium-dependent cell proliferation and inhibiting apoptosis (Bowen et al., 2013; Lehen'kyi et al., Am. J. Physiol., 2011, 301, C1281-9; Raphael et al., Proc. Natl. Acad. Sci. US A., 2014, 111, E3870-9). Thus, modulators of TRPV6 may provide novel therapeutic strategies for the treatment of TRPV6-expressing tumors (Bolanz et al., 2008; Bowen et al., 2013; Lehen'kyi et al., 2007; Schwarz et al., Cell Calcium, 2006, 39, 163-173). Any change in TRPV6 activity by a modulator would have devastating effects on the cell. Indeed, inhibition of TRPV6 channel activity or expression reduces calcium levels required for pro-survival and anti-apoptotic pathways by reducing calcineurin phosphatase activity (Roderick et al., Nat Rev Cancer., 2008, May;8(5):361-75). With regard to activation of TRPV6 channels, this leads to uncontrolled increase in calcium uptake, which overloads mitochondria and causes the release of cytochrome C, triggering the pro-apoptotic cascade (Bernardi et al., Subcell Biochem 2007, 45, 481-506).

[0006] Small molecule and peptide inhibitors of TRPV6 channel activity have been disclosed (TH-1177, Landowski et al., Pharm Res., 2011, 28, 322-30; soricidin or SOR-C13, Bowen et al., PLoS One. 2013, 8, e58866 and International PCT Application WO 2009 / 114943).

[0007] Several anti-TRPV6 polyclonal antibodies designed to recognize intracellular epitopes at the N-terminus or C-terminus have been disclosed and are commercially available (Lehen'kyi et al., 2007; Van der Eerden et al., J. Cell. Physiol., 2011, 227, 1951-1959; TRPV6 #C-16 (Santa Cruz); sc-31445 (Borthwick et al., Cell Calcium, 2008, 44, 147-57; Dhennin-Duthille et al., 2011); #ACC-036 (Alomone). The antibodies are used to detect TRPV6 and test for its correlation with cancer.

[0008] However, no anti-TRPV6 antibody, particularly an anti-TRPV6 monoclonal antibody, capable of specifically recognizing the extracellular epitope of the human TRPV6 channel has been reported so far, and no anti-TRPV6 antibody capable of regulating TRPV6 channel activity and thus inhibiting cancer proliferation has been reported so far. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2009 / 114943 [Non-patent literature]

[0010] [Non-Patent Document 1] Clapham et al., Nat. Rev. Neurosci., 2000, 2, 387-96 [Non-Patent Document 2] Hoenderop et al., Pflugers Arch. 2003, 446, 304-8 [Non-Patent Document 3] Saotome et al., Nature 2016, 534, 506-511 [Non-Patent Document 4] Pengら、Biochem. Biophys. Res. Commun., 2000, 278, 326-332

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[0011] The inventors have generated antibodies raised against extracellular epitopes of the TRPV6 channel, corresponding to the extracellular loop between the S1-S2 transmembrane domains and the extracellular part of the pore region (Figure 1). Such antibodies were able to modulate TRPV6 activity on the cell membrane, altering calcium influx currents and impairing calcium signaling, thereby inducing cancer cell apoptosis in vitro (Figures 7-11 and 13-15). It was shown that mouse monoclonal antibodies raised against epitopes at the pore level were able to inhibit tumor growth and the emergence of metastases in vivo in a human tumor xenograft mouse model (Figure 12). Such data open the perspective of therapeutic use of such anti-TRPV6 antibodies in diseases and disorders involving the TRPV6 channel, in particular diseases associated with TRPV6 expression, e.g., cancer.

[0012] The generated antibodies capable of detecting TRPV6 in cell membranes are useful in any in vitro or in vivo detection or diagnostic immunoassay on living, fixed or denatured cells or tissues, including but not limited to immunoblotting, immunoprecipitation, immunohistostaining, immunofluorescence, and immunohistochemistry, especially in clinic diagnosis using paraffin-embedded sections from patients suffering from various diseases and disorders involving the TRPV6 channel. For example, antibodies generated against epitopes in the extracellular portion of the extracellular loop or pore region between the S1-S2 transmembrane domains were able to detect TRPV6 expression in paraffin-embedded sections from prostate cancer resection specimens. At the same time, the antibodies did not generate any signal in paraffin-embedded sections from healthy prostate and benign prostatic hyperplasia specimens.

[0013] Thus, the present invention relates to an antibody against human transient receptor potential vanilloid 6 (TRPV6) channel protein, which binds to an extracellular epitope of the hTRPV6 protein of SEQ ID NO: 1, in particular a non-glycosylated epitope, for example an epitope derived from the first extracellular domain of human TRPV6 selected from any one of SEQ ID NOs: 3 to 5, 7, and 8, preferably any one of SEQ ID NOs: 3, 7, and 8, or an epitope derived from the third extracellular domain of human TRPV6 selected from SEQ ID NOs: 14 or 16. The antibody is preferably an antibody that regulates the activity of the human TRPV6 channel, preferably activates the human TRPV6 channel, and / or inhibits the proliferation of TRPV6-expressing cancer cells, preferably by inducing apoptosis of the cells.

[0014] In some embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable CDR comprising at least one, preferably all three, of VH-CDR1 of SEQ ID NO: 27, VH-CDR2 of SEQ ID NO: 28, and VH-CDR3 of SEQ ID NO: 29, or functional variants thereof, and a light chain variable CDR comprising at least one, preferably all three, of VL-CDR1 of SEQ ID NO: 17, VL-CDR2 of amino acid sequence LVS, and VL-CDR3 of SEQ ID NO: 18, or functional variants thereof.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: a) a heavy chain variable CDR comprising at least one, preferably all three, of the VH-CDR1 of SEQ ID NO: 49, the VH-CDR2 of SEQ ID NO: 50 and the VH-CDR3 of SEQ ID NO: 51, or functional variants thereof, and a light chain variable CDR comprising at least one, preferably all three, of the VL-CDR1 of SEQ ID NO: 38, the VL-CDR2 of the amino acid sequence WAS and the VL-CDR3 of SEQ ID NO: 39, or functional variants thereof, or b) A heavy chain variable CDR comprising at least one, preferably all three, of the VH-CDR1 of SEQ ID NO: 98, the VH-CDR2 of SEQ ID NO: 99, and the VH-CDR3 of SEQ ID NO: 100, or functional variants thereof, and a light chain variable CDR comprising at least one, preferably all three, of the VL-CDR1 of SEQ ID NO: 88, the VL-CDR2 of amino acid sequence SDS, and the VL-CDR3 of SEQ ID NO: 89, or functional variants thereof.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: a) a heavy chain variable CDR comprising at least one, preferably all three, of the VH-CDR1 of SEQ ID NO: 118, the VH-CDR2 of SEQ ID NO: 119, and the VH-CDR3 of SEQ ID NO: 120, or functional variants thereof, and a light chain variable CDR comprising at least one, preferably all three, of the VL-CDR1 of SEQ ID NO: 108, the VL-CDR2 of the amino acid sequence YDS, and the VL-CDR3 of SEQ ID NO: 109, or functional variants thereof, b) a heavy chain variable CDR comprising at least one, preferably all three, of the VH-CDR1 of SEQ ID NO: 138, the VH-CDR2 of SEQ ID NO: 139, and the VH-CDR3 of SEQ ID NO: 140, or functional variants thereof; and a light chain variable CDR comprising at least one, preferably all three, of the VL-CDR1 of SEQ ID NO: 128, the VL-CDR2 of the amino acid sequence QDS, and the VL-CDR3 of SEQ ID NO: 129, or functional variants thereof; c) a heavy chain variable CDR comprising at least one, preferably all three, of the VH-CDR1 of SEQ ID NO: 158, the VH-CDR2 of SEQ ID NO: 159 and the VH-CDR3 of SEQ ID NO: 160, or functional variants thereof, and a light chain variable CDR comprising at least one, preferably all three, of the VL-CDR1 of SEQ ID NO: 148, the VL-CDR2 of the amino acid sequence GDS and the VL-CDR3 of SEQ ID NO: 149, or functional variants thereof, or d) A heavy chain variable CDR comprising at least one, preferably all three, of VH-CDR1 of SEQ ID NO: 178, VH-CDR2 of SEQ ID NO: 179, and VH-CDR3 of SEQ ID NO: 180, or functional variants thereof, and a light chain variable CDR comprising at least one, preferably all three, of VL-CDR1 of SEQ ID NO: 168, VL-CDR2 of amino acid sequence YDS, and VL-CDR3 of SEQ ID NO: 169, or functional variants thereof.

[0017] In some preferred embodiments, the monoclonal antibody is a humanized monoclonal antibody comprising a heavy chain variable domain comprising VH-FR1 of SEQ ID NO: 73, VH-CDR1 of SEQ ID NO: 70, VH-FR2 of SEQ ID NO: 74, VH-CDR2 of SEQ ID NO: 71, VH-FR3 of SEQ ID NO: 75, VH-CDR3 of SEQ ID NO: 72, and VH-FR4 of SEQ ID NO: 76, or a functional variant thereof, and a light chain variable domain comprising VL-FR1 of SEQ ID NO: 61, VL-CDR1 of SEQ ID NO: 59, VL-FR2 of SEQ ID NO: 62, VL-CDR2 of amino acid sequence WAS, VL-FR3 of SEQ ID NO: 63, VL-CDR3 of SEQ ID NO: 60, and VL-FR4 of SEQ ID NO: 64 or 65, or a functional variant thereof.

[0018] In some embodiments, an antibody according to the invention comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have at least 90% identity with the sequence pair of SEQ ID NO: 34 and SEQ ID NO: 23 for the heavy chain variable domain sequence, respectively. In some preferred embodiments, the antibody comprises a heavy chain sequence and a light chain sequence that have at least 90% identity with the sequence pair of SEQ ID NO: 35 and SEQ ID NO: 24 for the heavy chain sequence and light chain sequence, respectively.

[0019] In some embodiments, an antibody according to the invention comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have at least 90% identity with any one of the following sequence pairs for the heavy chain variable domain sequence and the light chain variable domain sequence: SEQ ID NO:56 and SEQ ID NO:45 or 46; SEQ ID NO:77 and SEQ ID NO:66 or 67; SEQ ID NO:105 and SEQ ID NO:95 or 96, respectively, and preferably comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have at least 90% identity with any one of the following sequence pairs: SEQ ID NO:56 and SEQ ID NO:45 or 46; SEQ ID NO:77 and SEQ ID NO:66 or 67. In some preferred embodiments, the antibody comprises heavy and light chain sequences that have at least 90% identity to any one of the following sequence pairs for heavy and light chain sequences: SEQ ID NO:57 and SEQ ID NO:47; SEQ ID NO:78 or 80 and SEQ ID NO:68; SEQ ID NO:84 or 86 and SEQ ID NO:82; SEQ ID NO:106 or 107 and SEQ ID NO:97, respectively, and preferably comprises heavy chain variable domain sequences and light chain variable domain sequences that have at least 90% identity to sequence pairs selected from SEQ ID NO:57 and SEQ ID NO:47; SEQ ID NO:78 or 80 and SEQ ID NO:68; SEQ ID NO:84 or 86 and SEQ ID NO:82.

[0020] In some embodiments, an antibody according to the invention comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have at least 90% identity with any one of the following sequence pairs for the heavy chain variable domain sequence and the light chain variable domain sequence, respectively: SEQ ID NO: 125 and SEQ ID NO: 115 or 116; SEQ ID NO: 145 and SEQ ID NO: 135 or 136; SEQ ID NO: 165 and SEQ ID NO: 155 or 156; SEQ ID NO: 185 and SEQ ID NO: 175 or 176. In some preferred embodiments, the antibody comprises a heavy chain sequence and a light chain sequence that have at least 90% identity with any one of the following sequence pairs for the heavy chain sequence and the light chain sequence, respectively: SEQ ID NO: 126 or 127 and SEQ ID NO: 117; SEQ ID NO: 146 or 147 and SEQ ID NO: 137; SEQ ID NO: 166 or 167 and SEQ ID NO: 157; SEQ ID NO: 186 or 187 and SEQ ID NO: 177.

[0021] In some embodiments, the antibody according to the invention is a polyclonal or monoclonal antibody, in particular a recombinant, chimeric and / or humanized monoclonal antibody, preferably of the human IgG1 or IgG4 isotype.

[0022] In some embodiments, an antibody according to the invention is conjugated to a labeling agent or a therapeutic agent.

[0023] Another aspect of the present invention relates to an extracellular peptide antigen derived from human TRPV6 protein, comprising an sequence having at least 90% identity to any one of SEQ ID NOs: 3 to 5, 7, 8, 10, 14 or 16, which induces the production of antibodies according to the present disclosure.

[0024] Another aspect of the invention relates to an expression vector for the recombinant production of an antibody according to the present disclosure in a host cell, comprising at least one nucleic acid encoding the heavy and / or light chain of said antibody.

[0025] In some preferred embodiments, the expression vector comprises a nucleic acid sequence pair having at least 90% identity to the sequence pair of SEQ ID NO:37 and SEQ ID NO:26.

[0026] In some other preferred embodiments, the expression vector comprises a nucleic acid sequence pair having at least 90% identity to any one of the following sequence pairs: SEQ ID NO:48 and SEQ ID NO:58; SEQ ID NO:69 and SEQ ID NO:79; SEQ ID NO:69 and SEQ ID NO:81; SEQ ID NO:83 and SEQ ID NO:85; SEQ ID NO:83 and SEQ ID NO:87.

[0027] Another aspect of the invention relates to a pharmaceutical composition comprising at least an antibody according to the present disclosure and a pharma- ceutically acceptable solvent.

[0028] Another aspect of the invention relates to an antibody according to the present disclosure for use as a medicine, in particular for use in the treatment of a disease associated with TRPV6 expression, such as cancer, in particular cancer selected from the group consisting of endometrial cancer, leukemia, and breast, pancreatic, prostate, colon, ovarian and thyroid carcinoma, preferably prostate cancer.

[0029] Another aspect of the present invention relates to the use of an antibody according to the present disclosure for the in vitro diagnosis or prognosis of a disease associated with TRPV6 expression, such as cancer, in particular cancer selected from the group consisting of endometrial cancer, leukemia, and carcinoma of the breast, pancreas, prostate, colon, ovary, and thyroid, preferably prostate cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] antibody The present invention also relates to antibodies against the human transient receptor potential vanilloid 6 (TRPV6) channel protein that bind to an extracellular epitope of this protein.

[0031] As used herein, the term "antibody" refers to an "isolated antibody." Antibodies refer to glycoproteins produced by lymphoid cells in response to stimulation with an immunogen. Antibodies have the ability to react specifically and selectively in vitro and in vivo with the antigenic determinants or epitopes that elicit their production or antigenic determinants closely related to the homologous antigen.

[0032] The expressions "antibody recognizing antigen (X)", "antibody having specificity for antigen (X)", "anti-X antibody", "antibody to X" and "antibody to" are used interchangeably herein with the term "antibody that specifically binds to antigen (X)".

[0033] The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen-binding site typically contains six CDRs, including the CDRs of each of the heavy and light chain V regions. The framework region (FR) refers to the amino acid sequence inserted between the CDRs. Thus, the variable regions of the light and heavy chains typically contain four framework regions and three CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0034] Residues in antibody variable domains are conventionally numbered according to the scheme devised by Kabat et al., which is described in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter "Kabat et al."). This numbering scheme is used herein. The Kabat residue nomenclature does not always correspond directly to the linear numbering of amino acid residues in the sequence of SEQ ID NO:. The actual linear amino acid sequence, whether in the framework region or in the complementarity determining region (CDR) of the basic variable domain structure, may contain fewer or additional amino acids than the strict Kabat numbering, which corresponds to the shortening of or insertion into the structural element. The exact Kabat numbering of residues can be determined for a given antibody by alignment of homologous residues with the "standard" Kabat numbering sequence in the antibody sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2), and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2), and residues 89-97 (L-CDR3) according to the Kabat numbering system.

[0035] In the present invention, the terms "antibody" and "immunoglobulin" are equivalent and are used indiscriminately. Antibodies are designated as "Ab" and immunoglobulins as "Ig".

[0036] As used herein, the term "recombinant antibody" refers to an antibody that is generated, expressed, produced, or isolated by recombinant means, e.g., an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a recombinant combinatorial antibody library, an antibody isolated from an animal (e.g., a mouse) transgenic with human immunoglobulin genes, or an antibody generated, expressed, produced, or isolated by any other method that associates a particular immunoglobulin gene sequence (e.g., a human immunoglobulin gene sequence) with other DNA sequences. Recombinant antibodies include, for example, chimeric antibodies and humanized antibodies. In some embodiments, the recombinant human antibodies of the invention have an amino acid sequence identical to a naturally occurring human antibody, but are structurally different from naturally occurring human antibodies. For example, in some embodiments, the glycosylation pattern differs as a result of the recombinant production of the recombinant human antibody. In some embodiments, the recombinant human antibody is chemically modified by the addition or subtraction of at least one covalent chemical bond compared to the structure of a human antibody that occurs naturally in humans.

[0037] "Epitope" or antigenic determinant refers to that portion of an antigen that is recognized by an antibody.

[0038] As used herein, the term "identity" refers to the sequence similarity between two polypeptide molecules or two nucleic acid molecules. When a position in both sequences being compared is occupied by the same base or the same amino acid residue, the respective molecules are identical at that position. The percentage of identity between two sequences corresponds to the number of matching positions shared by the two sequences divided by the number of positions being compared, multiplied by 100. Generally, two sequences are compared when aligned to obtain maximum identity. This identity can be calculated by alignment, for example, using the pileup program of GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) or any of the sequence comparison algorithms, such as BLAST, FASTA, or CLUSTALW. In the following description, the standard one-letter amino acid code is used.

[0039] The antibody according to the present invention binds to an epitope located in the extracellular (EC) region of one of the human TRPV6 proteins. Human TRPV6 has the amino acid sequence of UniProtKB / Swiss-Prot NP_061116.5 or Q9H1D0.3 (SEQ ID NO: 1). The 3D crystal structure of TRPV6 has been determined (Saotome et al., Nature 2016, 534, 506-511).

[0040] TRPV6 has three extracellular (EC) regions. EC1 is located between the first transmembrane region (S1) and the second transmembrane region (S2), EC2 is located between the third transmembrane region (S3) and the fourth transmembrane region (S4), and the third extracellular region is divided into two subregions: EC3a located between the fifth transmembrane region (S5) and the intramembrane (IM) pore-forming region, and EC3b located between the intramembrane (IM) pore-forming region and the sixth transmembrane region (S6). Based on the structure prediction, EC1 is predicted to correspond to positions 389 to 425 of SEQ ID NO: 1, EC2 is predicted to correspond to positions 484 to 489 of SEQ ID NO: 1, EC3a is predicted to correspond to positions 563 to 565 of SEQ ID NO: 1, and EC3b is predicted to correspond to positions 586 to 596 of SEQ ID NO: 1. However, the location of the extracellular region of hTRPV6 that can be effectively accessed on the cell surface and that can be bound by antibodies may differ slightly from the predicted location. The TRPV6 extracellular region can be accurately determined using the 3D crystal structure of TRPV6 (Saotome et al., Nature 2016, 534, 506-511).

[0041] The epitope to which the antibody according to the invention binds may also comprise adjacent sequences (usually up to 5 amino acids; 1, 2, 3, 4 or 5 amino acids) from adjacent transmembrane (TM) and / or intramembrane (IM) regions. The epitope to which the antibody binds may be a variant of the wild-type human TRPV6 sequence that does not modify the specificity of the antibody for the wild-type human TRPV6 protein. This means that the variant epitope induces cross-reactive antibodies that bind with high affinity to both the variant epitope and the wild-type epitope.

[0042] The extracellular epitope to which the antibody of the present invention binds is preferably derived from the first or third extracellular domain of human TRPV6 (hTRPV6).

[0043] In certain embodiments, the extracellular epitope to which the antibody of the invention binds is not glycosylated. In particular, the epitope does not have a glycosylation site. The glycosylation site is preferably NXS or NXT, where X can be any amino acid.

[0044] In some embodiments, the antibody binds to an epitope from the first extracellular domain (EC1) of human TRPV6, derived from SEQ ID NO:2 (LLQEAYMTPKDDIRLVG); hTRPV6 residues 412-428 or hTRPV6 residues 412-428. In some preferred embodiments, the epitope is selected from the group consisting of SEQ ID NO:3-5, 7, 8, preferably SEQ ID NO:3, 7 or 8. SEQ ID NO:3 (QEAYMTPKDDIRLVG) corresponds to hTRPV6 residues 414-428, SEQ ID NO:4 (QEAYMTPKDDIR) corresponds to hTRPV6 residues 414-425, SEQ ID NO:5 (LLQEAYMTPKDDIR) corresponds to hTRPV6 residues 412-425,

[0045] [ka]

[0046] corresponds to hTRPV6 415-426, which has a D to E substitution at the 8th and 9th positions of the peptide sequence and an L to R substitution at the final position of the peptide sequence, and sequence number 8 (EAYMTPKDDIRL) corresponds to hTRPV6 415-426.

[0047] In some embodiments, the antibody binds to an epitope derived from the third extracellular domain (EC3) of human TRPV6. This epitope may be derived from EC3a, in particular the sequence of SEQ ID NO: 9 (IFQTEDPEELGHFYDYPMALFST; hTRPV6 551-573), or may be derived from EC3b. In some preferred embodiments, the epitope is selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO: 16. SEQ ID NO: 14 (TEDPEELGHFYDYPMA) corresponds to hTRPV6 554-569, and SEQ ID NO: 16 (DGPANYNVDLPFMYS) corresponds to hTRPV6 582-596.

[0048] The antibody according to the present invention specifically recognizes human TRPV6 protein on cell membrane, particularly human mature glycosylated TRPV6 protein, which means that the antibody has a relatively high affinity for the epitope of TRPV6, but does not substantially recognize it, and also binds to peptides other than the peptide of interest.

[0049] As used herein, the term "relatively high affinity" refers to a binding affinity of at least 10 -6 M, preferably at least about 10 -7 M, and even more preferably 10 -8 M~10 -10 M refers to the binding affinity between the antibody and the protein of interest. Such affinity determination is preferably carried out under standard competitive binding immunoassay conditions, which are common knowledge to those skilled in the art.

[0050] In some embodiments, the antibody modulates the activity of the human TRPV6 channel. In certain embodiments, the antibody activates the human TRPV6 channel. In another specific embodiment, the antibody inhibits the human TRPV6 channel.

[0051] Modulation of TRPV6 channel activity by the antibodies of the present invention may be quantified according to standard techniques well known in the art, such as those disclosed in the Examples herein, including, but not limited to, the whole-cell patch clamp technique; the store-operated calcium entry (SOCE) assay as disclosed in Raphael et al., 2014; the radioactive uptake assay measuring ion transport across the ion channel as disclosed in Nimigean CM, Nat Protoc. 2006; 1(3):1207-12, and others.

[0052] In some embodiments, the antibody inhibits the proliferation of TRPV6 expressing cells, particularly cancer cells. Advantageously, the antibody induces apoptosis in TRPV6 expressing cells, such as cancer cells. The inhibition of proliferation or induction of apoptosis in TRPV6 expressing cells by the antibody of the present invention can be quantified according to standard techniques well known in the art, such as those disclosed in the Examples herein. For example, the inhibition of proliferation can be measured using cell viability assays using MTS or MTT; cell cycle assays, cell counting assays, LDH leakage, total cell protein measurement, neutral red, Alamar Blue®, uridine incorporation assays, or by the expression of Ki-67, PCNA, CdK4, and cyclin D proteins in Western blot / IHC. Induction of apoptosis may be measured using TUNEL assay, Hoechst staining, detection of apoptotic markers such as phosphatidylserine exposure, activation of caspases, calpains, and cathepsins, changes in mitochondrial membrane potential, or cell membrane blebbing and nuclear condensation, DNA ladder assay, cleaved caspase 3 assay, or Annexin V binding.

[0053] The antibody according to the invention may comprise a whole antibody or an antigen-binding fragment thereof. The antibody fragment may be selected from the group consisting of Fv, ScFv, Sc(Fv)2, DsFv, Fab, F(ab)2, Fab' fragment, diabody, and single domain antibody (VHH). The variable region of the antibody according to the invention may be combined with a constant region domain, such as an IgA, IgM, IgE, IgG or IgD domain, in particular a human constant region domain; preferably an IgG, in particular a human IgG1 or IgG4 constant domain. Such constant regions may be further mutated or modified by methods known in the art, in particular to modify their binding ability to Fc receptors or to enhance the antibody half-life. The antibody may be glycosylated or non-glycosylated.

[0054] The antibody may be a monoclonal or polyclonal antibody, a non-recombinant or recombinant antibody, a chimeric or humanized antibody. A monoclonal antibody is a monospecific and bivalent immunoglobulin molecule. The term "antibody" is intended to include aggregates, polymers, derivatives, or conjugates of antibodies or antibody fragments. Examples of derivatives include variants and constructs using antigen-binding fragments of such antibodies, e.g., multivalent and / or multispecific antibodies.

[0055] In some embodiments, the antibody is a polyclonal antibody, for example a rabbit polyclonal antibody. The polyclonal antibody according to the present invention is a monospecific antibody, which means that the polyclonal antibody is specific for an extracellular epitope of hTRPV6 protein. In general, such a polyclonal antibody is obtained by immunization with a peptide having the extracellular epitope sequence or a closely related sequence that induces cross-reactive antibodies as defined above. In some preferred embodiments, the polyclonal antibody binds to the epitope of SEQ ID NO:3.

[0056] In some embodiments, the antibody is a monoclonal antibody (mAb), preferably a human, humanized or chimeric monoclonal antibody. A chimeric antibody has human constant domains and variable domains from a non-human source, typically a mouse (human / mouse chimeric antibody). The monoclonal antibody is preferably a recombinant antibody.

[0057] In certain embodiments, a monoclonal antibody or antigen-binding fragment thereof that binds to the epitope of SEQ ID NO:8 and its variant epitope of SEQ ID NO:7 comprises a heavy chain variable CDR comprising at least one, preferably all three, of VH-CDR1 of SEQ ID NO:27, VH-CDR2 of SEQ ID NO:28, and VH-CDR3 of SEQ ID NO:29, or functional variants thereof, and a light chain variable CDR comprising at least one, preferably all three, of VL-CDR1 of SEQ ID NO:17, VL-CDR2 of amino acid sequence LVS, and VL-CDR3 of SEQ ID NO:18, or functional variants thereof. Preferably, the monoclonal antibody or antigen-binding fragment thereof, which binds to the epitope of SEQ ID NO:8 and its variant epitope of SEQ ID NO:7, comprises a heavy chain variable domain comprising at least one, preferably all three, of VH-CDR1 of SEQ ID NO:27, VH-CDR2 of SEQ ID NO:28, and VH-CDR3 of SEQ ID NO:29, or functional variants thereof, and a heavy chain variable domain and a light chain variable domain selected from VL-CDR1 of SEQ ID NO:17, VL-CDR2 of amino acid sequence LVS, and VL-CDR3 of SEQ ID NO:18, or at least one, preferably all three, of functional variants thereof.

[0058] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof that binds to the epitope of SEQ ID NO: 14 is a) a heavy chain variable CDR comprising at least one, preferably all three, of the VH-CDR1 of SEQ ID NO: 49, the VH-CDR2 of SEQ ID NO: 50 and the VH-CDR3 of SEQ ID NO: 51, or functional variants thereof, and a light chain variable CDR comprising at least one, preferably all three, of the VL-CDR1 of SEQ ID NO: 38, the VL-CDR2 of the amino acid sequence WAS and the VL-CDR3 of SEQ ID NO: 39, or functional variants thereof, or b) a heavy chain variable CDR comprising at least one, preferably all three, of SEQ ID NO: 98 VH-CDR1, SEQ ID NO: 99 VH-CDR2 and SEQ ID NO: 100 VH-CDR3 or functional variants thereof, and a light chain variable CDR comprising at least one, preferably all three, of SEQ ID NO: 88 VL-CDR1, SEQ ID NO: 88 VL-CDR2 of amino acid sequence SDS and SEQ ID NO: 89 VL-CDR3 or functional variants thereof; Includes.

[0059] In some preferred embodiments, a monoclonal antibody or antigen-binding fragment thereof that binds to the epitope of SEQ ID NO: 14 comprises a heavy chain variable domain comprising at least one, preferably all three, of VH-CDR1 of SEQ ID NO: 49, VH-CDR2 of SEQ ID NO: 50, and VH-CDR3 of SEQ ID NO: 51, or functional variants thereof, and a light chain variable domain comprising at least one, preferably all three, of VL-CDR1 of SEQ ID NO: 38, VL-CDR2 of amino acid sequence WAS, and VL-CDR3 of SEQ ID NO: 39, or functional variants thereof.

[0060] In some other embodiments, the monoclonal antibody or antigen-binding fragment thereof that binds to the epitope of SEQ ID NO: 14 comprises a heavy chain variable domain comprising at least one, preferably all three, of a VH-CDR1 of SEQ ID NO: 98, a VH-CDR2 of SEQ ID NO: 99, and a VH-CDR3 of SEQ ID NO: 100, or functional variants thereof, and a light chain variable domain comprising at least one, preferably all three, of a VL-CDR1 of SEQ ID NO: 88, a VL-CDR2 of amino acid sequence SDS, and a VL-CDR3 of SEQ ID NO: 89, or functional variants thereof.

[0061] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof that binds to the epitope of SEQ ID NO: 16 is a) a heavy chain variable CDR comprising at least one, preferably all three, of the VH-CDR1 of SEQ ID NO: 118, the VH-CDR2 of SEQ ID NO: 119, and the VH-CDR3 of SEQ ID NO: 120, or functional variants thereof, and a light chain variable CDR comprising at least one, preferably all three, of the VL-CDR1 of SEQ ID NO: 108, the VL-CDR2 of the amino acid sequence YDS, and the VL-CDR3 of SEQ ID NO: 109, or functional variants thereof, b) a heavy chain variable CDR comprising at least one, preferably all three, of the VH-CDR1 of SEQ ID NO: 138, the VH-CDR2 of SEQ ID NO: 139, and the VH-CDR3 of SEQ ID NO: 140, or functional variants thereof; and a light chain variable CDR comprising at least one, preferably all three, of the VL-CDR1 of SEQ ID NO: 128, the VL-CDR2 of the amino acid sequence QDS, and the VL-CDR3 of SEQ ID NO: 129, or functional variants thereof; c) a heavy chain variable CDR comprising at least one, preferably all three, of the VH-CDR1 of SEQ ID NO: 158, the VH-CDR2 of SEQ ID NO: 159 and the VH-CDR3 of SEQ ID NO: 160, or functional variants thereof, and a light chain variable CDR comprising at least one, preferably all three, of the VL-CDR1 of SEQ ID NO: 148, the VL-CDR2 of the amino acid sequence GDS and the VL-CDR3 of SEQ ID NO: 149, or functional variants thereof, or d) a heavy chain variable CDR comprising at least one, preferably all three, of the VH-CDR1 of SEQ ID NO: 178, the VH-CDR2 of SEQ ID NO: 179, and the VH-CDR3 of SEQ ID NO: 180, or functional variants thereof; and a light chain variable CDR comprising at least one, preferably all three, of the VL-CDR1 of SEQ ID NO: 168, the VL-CDR2 of the amino acid sequence YDS, and the VL-CDR3 of SEQ ID NO: 169, or functional variants thereof; Includes.

[0062] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof that binds to the epitope of SEQ ID NO: 16 is a) a heavy chain variable domain comprising at least one, preferably all three, of the VH-CDR1 of SEQ ID NO: 118, the VH-CDR2 of SEQ ID NO: 119 and the VH-CDR3 of SEQ ID NO: 120, or functional variants thereof, and a light chain variable domain comprising at least one, preferably all three, of the VL-CDR1 of SEQ ID NO: 108, the VL-CDR2 of the amino acid sequence YDS and the VL-CDR3 of SEQ ID NO: 109, or functional variants thereof, b) a heavy chain variable domain comprising at least one, preferably all three, of the VH-CDR1 of SEQ ID NO: 138, the VH-CDR2 of SEQ ID NO: 139 and the VH-CDR3 of SEQ ID NO: 140, or functional variants thereof, and a light chain variable domain comprising at least one, preferably all three, of the VL-CDR1 of SEQ ID NO: 128, the VL-CDR2 of the amino acid sequence QDS and the VL-CDR3 of SEQ ID NO: 129, or functional variants thereof, c) a heavy chain variable domain comprising at least one, preferably all three, of the VH-CDR1 of SEQ ID NO: 158, the VH-CDR2 of SEQ ID NO: 159 and the VH-CDR3 of SEQ ID NO: 160, or functional variants thereof; and a light chain variable domain comprising at least one, preferably all three, of the VL-CDR1 of SEQ ID NO: 148, the VL-CDR2 of the amino acid sequence GDS and the VL-CDR3 of SEQ ID NO: 149, or functional variants thereof; and d) a heavy chain variable domain comprising at least one, preferably all three, of the VH-CDR1 of SEQ ID NO: 178, the VH-CDR2 of SEQ ID NO: 179, and the VH-CDR3 of SEQ ID NO: 180, or functional variants thereof; and a light chain variable domain comprising at least one, preferably all three, of the VL-CDR1 of SEQ ID NO: 168, the VL-CDR2 of the amino acid sequence YDS, and the VL-CDR3 of SEQ ID NO: 169, or functional variants thereof. The invention comprises a heavy chain variable domain and a light chain variable domain selected from:

[0063] As used herein, "functional variant" with respect to variants of antibody sequences (CDR, FR or other) means that the antibody comprising the sequence variant specifically recognizes human TRPV6 protein. It is intended that the monoclonal antibody or antigen-binding fragment thereof may have one, two, three, four, five, six or more changes in the amino acid sequence of one, two, three, four, five or six CDRs of the monoclonal antibody provided herein. It is intended that the first, second, third, fourth, fifth, sixth, seventh, eighth, nineth, tenth, eleventh, twelfth or thirteenth amino acid of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 of the VJ or VDJ region of the light or heavy chain variable region of the antibody may have an insertion, deletion or substitution with a conservative or non-conservative amino acid. Such amino acids that may be substituted or constitute this substitution are disclosed below. In some specific embodiments, the monoclonal antibody or antigen-binding fragment has one or two conservative substitutions in the amino acid sequence of one, two, three, four, five or six CDRs of the monoclonal antibody provided herein. It is also contemplated that the monoclonal antibody or antigen-binding fragment thereof may have one, two, three, four, five, six, seven, eight, nine, ten or more changes in the amino acid sequence of one, two, three, four, five, six, seven, eight FRs of the monoclonal antibody provided herein. It is contemplated that the FR sequence has insertions, deletions or substitutions with conservative or non-conservative amino acids. Such amino acids that may be substituted or constitute the substitution are disclosed above. In some specific embodiments, the monoclonal antibody or antigen-binding fragment thereof has one, two, three, four, five, preferably one or two conservative substitutions in the amino acid sequence of one, two, three, four, five, six, seven, eight FRs of the monoclonal antibody provided herein.

[0064] In some embodiments, the substitution is a conservative substitution, i.e., a substitution of one amino acid with another amino acid having similar chemical or physical properties (size, charge or polarity), and this substitution generally does not adversely affect the biochemical, biophysical and / or biological properties of the antibody. In particular, this substitution does not disrupt the interaction of the antibody with human TRPV6 protein. This conservative substitution is advantageously selected from one of the following five groups: group 1-small aliphatic non-polar or slightly polar residues (A, S, T, P, G); group 2-polar negatively charged residues and their amides (D, N, E, Q); group 3-polar positively charged residues (H, R, K); group 4-large aliphatic non-polar residues (M, L, I, V, C); and group 5-large aromatic residues (F, Y, W).

[0065] In some embodiments, the antibody is a monoclonal antibody that binds to an epitope bound by an antibody having the six VH-CDR sequences and the six VL-CDR sequences defined above.

[0066] In some preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable CDR comprising at least one, preferably all three, of VH-CDR1 of SEQ ID NO: 49, VH-CDR2 of SEQ ID NO: 50, and VH-CDR3 of SEQ ID NO: 51, or functional variants thereof, and a light chain variable CDR comprising at least one, preferably all three, of VL-CDR1 of SEQ ID NO: 38, VL-CDR2 of amino acid sequence WAS, and VL-CDR3 of SEQ ID NO: 39, or functional variants thereof. Preferably, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising at least one, preferably all three, of VH-CDR1 of SEQ ID NO: 49, VH-CDR2 of SEQ ID NO: 50, and VH-CDR3 of SEQ ID NO: 51, or functional variants thereof, and a light chain variable domain comprising VL-CDR1 of SEQ ID NO: 38, VL-CDR2 of amino acid sequence WAS, and VL-CDR3 of SEQ ID NO: 39, or at least one, preferably all three, functional variants thereof.

[0067] In some further preferred embodiments, the antibody is a humanized monoclonal antibody or antigen-binding fragment thereof that binds to the epitope of SEQ ID NO: 14 and comprises a heavy chain variable domain comprising VH-FR1 of SEQ ID NO: 73, VH-CDR1 of SEQ ID NO: 70, VH-FR2 of SEQ ID NO: 74, VH-CDR2 of SEQ ID NO: 71, VH-FR3 of SEQ ID NO: 75, VH-CDR3 of SEQ ID NO: 72, and VH-FR4 of SEQ ID NO: 76, or a functional variant thereof, and a light chain variable domain comprising VL-FR1 of SEQ ID NO: 61, VL-CDR1 of SEQ ID NO: 59, VL-FR2 of SEQ ID NO: 62, VL-CDR2 of amino acid sequence WAS, VL-FR3 of SEQ ID NO: 63, VL-CDR3 of SEQ ID NO: 60, and VL-FR4 of SEQ ID NO: 64 or 65, or a functional variant thereof.

[0068] In certain embodiments, an antibody or antigen-binding fragment thereof according to the present disclosure comprises a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 34, and a light chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 23.

[0069] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present disclosure comprises: a) a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 56, and a light chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 45 or 46; b) a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 77, and a light chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 66 or 67; c) a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 105, and a light chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 95 or 96; d) a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 125, and a light chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 115 or 116; e) a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 145, and a light chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 135 or 136; f) a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 165, and a light chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 155 or 156; and g) a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 185, and a light chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 175 or 176. The invention comprises a heavy chain variable domain and a light chain variable domain selected from:

[0070] In some preferred embodiments, an antibody or antigen-binding fragment thereof according to the present disclosure comprises a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 56 and a light chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 45 or 46, or a heavy chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 77 and a light chain variable domain comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 66 or 67.

[0071] In some more specific embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence having at least 90% identity to SEQ ID NO:35 and a light chain amino acid sequence having at least 90% identity to SEQ ID NO:24.

[0072] In some more specific embodiments, the antibody or antigen-binding fragment thereof comprises: a) a heavy chain amino acid sequence having at least 90% identity to SEQ ID NO:57, and a light chain amino acid sequence having at least 90% identity to SEQ ID NO:47; b) a heavy chain amino acid sequence having at least 90% identity to SEQ ID NO: 78 or 80, and a light chain amino acid sequence having at least 90% identity to SEQ ID NO: 68; c) a heavy chain amino acid sequence having at least 90% identity to SEQ ID NO: 84 or 86, and a light chain amino acid sequence having at least 90% identity to SEQ ID NO: 82; d) a heavy chain amino acid sequence having at least 90% identity to SEQ ID NO: 106 or 107, and a light chain amino acid sequence having at least 90% identity to SEQ ID NO: 97; e) a heavy chain amino acid sequence having at least 90% identity to SEQ ID NO: 126 or 127, and a light chain amino acid sequence having at least 90% identity to SEQ ID NO: 117; f) a heavy chain amino acid sequence having at least 90% identity to SEQ ID NO: 146 or 147, and a light chain amino acid sequence having at least 90% identity to SEQ ID NO: 137; g) a heavy chain amino acid sequence having at least 90% identity to SEQ ID NO: 166 or 167, and a light chain amino acid sequence having at least 90% identity to SEQ ID NO: 157; or h) a heavy chain amino acid sequence having at least 90% identity to SEQ ID NO: 186 or 187, and a light chain amino acid sequence having at least 90% identity to SEQ ID NO: 177; Includes.

[0073] In some preferred embodiments, an antibody or antigen-binding fragment thereof according to the present disclosure has a heavy chain amino acid sequence and a light chain amino acid sequence that have at least 90% identity to any one of the following sequence pairs: SEQ ID NO:47 and SEQ ID NO:57, SEQ ID NO:68 and SEQ ID NO:78, SEQ ID NO:68 and SEQ ID NO:80, SEQ ID NO:82 and SEQ ID NO:84, and SEQ ID NO:82 and SEQ ID NO:86.

[0074] In some embodiments, the antibodies of the invention comprise a sequence provided in Table I.

[0075] The antibodies of the invention can be produced by conventional techniques known to those skilled in the art. For example, monoclonal antibodies are produced from hybridomas obtained by fusion of B lymphocytes of animals immunized with CEA antigen with myelomas according to the technique of Kohler and Milstein (Nature, 1975, 256, 495-497), which hybridomas are cultivated in vitro, in particular in fermenters. Chimeric and / or humanized recombinant antibodies and antibody fragments can be prepared from hybridoma cells specific for the antigen by conventional techniques of recombinant DNA cloning and expression. Human antibodies can be obtained from transgenic mice carrying human immunoglobulin loci.

[0076] In some embodiments, the antibody is modified. In particular, the antibody constant region may be mutated or modified by methods known in the art, in particular to modify their binding ability to Fc receptors, to enhance antibody half-life, or to conjugate to a desired agent, such as a labeling agent or a therapeutic agent. For example, covalent binding of an agent to an antibody may be achieved by incorporating a reactive group into the antibody and then using this group to covalently bind the agent. Alternatively, covalent binding may be achieved by modifying a fusion protein.

[0077] In some embodiments, the antibody is conjugated with a labeling agent. The labeling agent is any agent that generates a detectable and / or quantifiable signal, in particular a radioactive, magnetic or luminescent (radioluminescent, chemiluminescent, bioluminescent, fluorescent or phosphorescent) agent. The antibody may be directly or indirectly labeled via covalent or non-covalent attachment using standard conjugation techniques well known to those skilled in the art. Directly detectable labels include radioisotopes and fluorophores. Indirectly detectable labels are detected by labeling with an additional reagent that allows for detection. Indirectly detectable labels include, for example, chemiluminescent agents, enzymes that generate a visible or colored reagent formulation, and ligand binding partners that allow for the detection of a ligand (hapten, antibody, antigen, biotin) that can be detected by binding the ligand to a labeled ligand-specific binding partner. The detectable label according to the present invention may be any type of label, in particular a fluorophore, such as fluorescein or luciferase; a radioisotope, in particular a radioisotope suitable for scintigraphy, such as 99m Tc; or an enzyme, for example, horseradish peroxidase.

[0078] In some other embodiments, the antibody is conjugated to a drug, for example an anti-cancer drug.

[0079] The antibodies according to the invention are used for diagnostic and therapeutic purposes to target cells expressing TRPV6, in particular tumor cells. The target cells preferably highly express TRPV6.

[0080] In the present invention, "cells highly expressing TRPV6", particularly "tumor or cancer cells highly expressing TRPV6", refer to cells, e.g., tumor or cancer cells, that exhibit significantly higher expression levels of TRPV6 compared to the levels of normal cells of the corresponding tissue or organ in healthy individuals. TRPV6 expression levels are measured by standard gene expression assays based on quantitative analysis of mRNA (RT-PCR and others) or protein (immunoassays, e.g., ELISA and others).

[0081] The invention encompasses the use of mixtures or combinations of antibodies, for example a mixture of different anti-TRPV6 antibodies according to the invention, or a mixture of an antibody according to the invention and another antibody.

[0082] "A," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, the terms "a" (or "an"), "one or more," or "at least one" can be used interchangeably herein.

[0083] Peptide antigens for antibody generation and uses The present invention relates to extracellular peptide antigens derived from the human TRPV6 protein that induce the production of antibodies according to the invention.

[0084] The peptides of the present invention are isolated, recombinant, or synthetic peptides derived from the human Transient Receptor Potential Vanilloid 6 (TRPV6) protein and are distinct from the TRPV6 protein.

[0085] The peptide of the present invention is an extracellular peptide derived from one of the extracellular (EC) regions of the human TRPV6 protein defined above. The peptide according to the present invention may also comprise adjacent sequences (usually up to 5 amino acids; 1, 2, 3, 4 or 5 amino acids) derived from the adjacent transmembrane (TM) and / or intramembrane (IM) regions. The peptide of the present invention is preferably derived from the first or third extracellular region of human TRPV6.

[0086] The peptides of the invention are antigenic peptides, which means that immunization of a non-human mammal, for example a mouse or a rabbit, with a peptide according to the invention induces the production of antibodies according to the invention.

[0087] In some embodiments, the peptide is derived from the first extracellular domain (EC1) of human TRPV6, in particular the sequence of SEQ ID NO: 2 (LLQEAYMTPKDDIRLVG); hTRPV6 412-428 or hTRPV6 412-428. In some preferred embodiments, the peptide comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 7 or 8 and a sequence having at least 70% amino acid identity with any one of said sequences, preferably SEQ ID NO: 3, 7 or 8. SEQ ID NO: 3 (QEAYMTPKDDIRLVG) corresponds to hTRPV6 414-428, SEQ ID NO: 4 (QEAYMTPKDDIR) corresponds to hTRPV6 414-425, SEQ ID NO: 5 (LLQEAYMTPKDDIR) corresponds to hTRPV6 412-425,

[0088] [ka]

[0089] corresponds to hTRPV6 415-426, which has a D to E substitution at the 8th and 9th positions of the peptide sequence and an L to R substitution at the final position of the peptide sequence, and sequence number 8 (EAYMTPKDDIRL) corresponds to hTRPV6 415-426.

[0090] In some embodiments, the peptide is derived from the third extracellular domain (EC3) of human TRPV6. The peptide may be derived from the sequence of EC3a, in particular SEQ ID NO: 9 (IFQTEDPEELGHFYDYPMALFST; hTRPV6 551-573), or may be derived from EC3b. In some preferred embodiments, the peptide comprises or consists of a sequence selected from the group consisting of the sequences of SEQ ID NO: 14 and SEQ ID NO: 16, and sequences having at least 70% amino acid identity with any one of these sequences. SEQ ID NO: 14 (TEDPEELGHFYDYPMA) corresponds to hTRPV6 554-569. SEQ ID NO: 16 (DGPANYNVDLPFMYS) corresponds to hTRPV6 582-596. Preferably, the peptide has at least 75%, 80%, 85%, 87%, 90%, 95%, 98% or 99% identity with any one of these sequences. More preferably, the peptide has at least 95%, 98% or 99% identity to any one of these sequences.

[0091] The peptides typically consist of a sequence of up to 50 amino acids, preferably 20, 25, 30, 35, 40, 45 amino acids, derived from human TRPV6.

[0092] The present invention encompasses peptides comprising or consisting of a chain of naturally occurring amino acids (the 20 genetically encoded amino acids (A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, X, and Y) in the L and / or D configuration) linked via peptide bonds, and further includes peptidomimetics of peptides in which the amino acids and / or peptide bonds have been replaced by functional analogues. Such functional analogues include any known amino acid outside the 20 genetically encoded amino acids.

[0093] The present invention also encompasses modified peptides derived from this peptide by the introduction of any chemical modification at one or more amino acid residues, at the peptide bond, at the N-terminus and / or at the C-terminus of the peptide, as long as the modified peptide is functional. Such modifications, introduced into the peptide by conventional methods known to those skilled in the art, include, but are not limited to, the replacement of natural amino acids with non-proteinogenic amino acids (D-amino acids or amino acid analogs), the modification of the peptide bond, in particular with a bond of the retro or retro-inverso type or a bond different from the peptide bond, cyclization, and the addition of chemical groups to the side chain or terminus of the peptide, in particular for conjugating a drug of interest to the peptide of the invention. Such modifications may be used to increase antigenicity, immunogenicity, and / or bioavailability, or to label the peptide.

[0094] In some embodiments, the peptide is conjugated to a carrier protein to enhance the immunogenicity of the peptide. The peptide of the present invention can be conjugated to any carrier protein used in preparing antibodies. In some specific embodiments, the peptide is conjugated to KLH protein, for example, via peptide N-ter. In some preferred embodiments, the peptide is any one of SEQ ID NO: 3, 4, 5, 7 or 8 conjugated to a carrier protein, for example, KLH, via peptide N-ter.

[0095] In some embodiments, the peptide comprises an additional amino acid residue, in particular a lysine, at the N-ter or C-ter. For example, the peptide is SEQ ID NO:6 or SEQ ID NO:15. In some other embodiments, the peptide comprises a spacer sequence at the N-ter or C-ter. Such modifications are useful for conjugating an agent of interest to the peptide of the invention.

[0096] The peptides according to the invention are prepared by conventional techniques known to those skilled in the art, in particular by solid- or liquid-phase synthesis or by expression of recombinant DNA in suitable cell systems (eukaryotic or prokaryotic). The peptides are usually synthesized in solid phase according to the Fmoc technique first described by Merrifield et al. (J. Am. Chem. Soc., 1964, 85: 2149-) and purified by reversed-phase high-performance liquid chromatography.

[0097] The peptides according to the invention are used for the production of antibodies according to the invention.

[0098] The present invention therefore relates to the use of a peptide according to the invention for the generation of an antibody according to the invention.

[0099] The present invention also provides a method for producing a semiconductor device comprising the steps of: a) immunizing a non-human mammal, such as a laboratory rodent, such as a rabbit or mouse, with a peptide according to the invention to induce the production of anti-TRPV6 antibodies by B cells of this mammal; b) harvesting the anti-TRPV6 antibody or B cells from the mammal The present invention also relates to a method for producing an anti-TRPV6 antibody according to the present invention, comprising:

[0100] The immunization step is carried out according to standard protocols known in the art, for example, by use of the peptide coupled to a carrier, such as KLH protein.

[0101] Antibodies may be recovered from the serum of the immunized mammal. B cells may be isolated from the spleen of the immunized mammal.

[0102] The method may further comprise immortalizing the B cells, for example by fusing the B cells with a myeloma cell line, a lymphoblastoid cell line, a lymphoma cell or a heteromyeloma cell line, according to standard hybridoma production techniques. Preferably, the B cells are immortalized by fusion with a mouse myeloma cell line, more preferably a mouse myeloma cell line, for example the SP2 / 0 cell line, which does not produce any mouse antibodies and is immortalized and has the entire secretory machinery required for secreting immunoglobulins. The immortalized B cells are screened for the production of specific antibodies using conventional assays, for example ELISA. After screening, they are usually cloned using standard methods. The antibodies secreted by the immortalized B cells are recovered from the extracellular medium and usually further purified by conventional techniques known to those skilled in the art, for example affinity chromatography. Alternatively, the VH and VL fragments of the anti-TRPV6 antibody can be cloned from the B cells producing the anti-TRPV6 antibody according to the invention, and recombinant antibodies can be produced according to standard techniques well known in the art.

[0103] The anti-TRPV6 antibodies according to the present invention can also be produced by screening a phage display library. In particular, the VH and VL fragments of the anti-TRPV6 antibodies can also be screened from a phage display library using the peptide antigens according to the present invention, and recombinant antibodies can be produced according to standard techniques well known in the art.

[0104] The present invention also encompasses antibodies obtainable or accessible by the method of producing an anti-TRPV6 antibody according to the present invention.

[0105] Polynucleotides and Vectors The present invention also relates to isolated polynucleotides encoding, in an expressible form, the antibodies of the present invention.

[0106] A polynucleotide encoding an antibody in an expressible form refers to a nucleic acid molecule that upon expression in a cell or cell-free system produces a functional peptide or antibody.

[0107] The polynucleotide, either synthetic or recombinant, can be single-stranded and / or double-stranded, DNA, RNA, or a combination thereof. The polynucleotide is operably linked to at least one transcriptional regulatory sequence, and optionally, at least one translational regulatory sequence. Preferably, the polynucleotide contains a coding sequence optimized for the host in which the peptide or antibody is expressed.

[0108] In some embodiments, the polynucleotide encodes at least the VH and / or VL domains of a monoclonal antibody according to the invention. In some preferred embodiments, the polynucleotide encoding the VH domain comprises a sequence having at least 80% identity with SEQ ID NO: 36, and the polynucleotide encoding the VL domain comprises a sequence having at least 80% identity with SEQ ID NO: 25. In some specific embodiments, the polynucleotide encodes the heavy and / or light chains of an antibody according to the present disclosure. In some preferred embodiments, the heavy and light chains of an antibody according to the present disclosure are encoded by at least one polynucleotide comprising a sequence pair selected from the group consisting of SEQ ID NO: 37 and SEQ ID NO: 26, and a sequence having at least 80% identity with the aforementioned sequences.

[0109] In some other preferred embodiments, the heavy and light chains of the antibody according to the present disclosure are encoded by at least one polynucleotide comprising a pair of sequences selected from the group consisting of SEQ ID NO: 48 and SEQ ID NO: 58, SEQ ID NO: 69 and SEQ ID NO: 79, SEQ ID NO: 69 and SEQ ID NO: 81, SEQ ID NO: 83 and SEQ ID NO: 85, SEQ ID NO: 83 and SEQ ID NO: 87, and sequences having at least 80% identity with the foregoing sequences. The polynucleotide may comprise or consist of a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequences disclosed above.

[0110] Polynucleotides according to the invention are prepared by conventional methods known in the art, for example by amplification of nucleic acid sequences by PCR or RT-PCR, by screening of genomic DNA libraries by hybridization with homologous probes, or by other methods by total or partial chemical synthesis.

[0111] Another aspect of the present invention is a recombinant vector comprising this polynucleotide, preferably comprising a pair of polynucleotide sequences encoding at least the VH and / or VL domains of the monoclonal antibody according to the invention as defined above. The recombinant vector is advantageously an expression vector capable of expressing this polynucleotide when delivered into a host cell, such as a prokaryotic or eukaryotic cell, such as a mammalian or bacterial cell. Recombinant vectors include conventional vectors used in gene modification, vaccines, and gene therapy, including, for example, plasmids and viral vectors.

[0112] Recombinant vectors are constructed and introduced into host cells by conventional recombinant DNA and genetic engineering techniques known in the art.

[0113] Thus, according to a further aspect of the present invention there is provided a host cell transformed with this polynucleotide or recombinant vector.

[0114] The polynucleotides, vectors and cells of the invention are useful for producing an antibody of the invention using well-known recombinant DNA techniques.

[0115] Pharmaceutical Compositions and Therapeutic Uses The present invention also relates to a pharmaceutical composition comprising, as active substance, at least one antibody, polynucleotide and / or vector according to the invention, together with at least one pharma- ceutically acceptable solvent.

[0116] Pharmaceutical compositions are formulated for administration by a number of routes, including but not limited to oral, parenteral, and topical. Pharmaceutical solvents are those appropriate for the planned route of administration and are well known in the art.

[0117] The pharmaceutical composition comprises a therapeutically effective amount of the antibody / polynucleotide / vector sufficient to show a positive medical response in the individual to whom it is administered. A positive medical response refers to a reduction in subsequent (prophylactic treatment) or established (therapeutic treatment) disease symptoms. A positive medical response includes a partial or total inhibition of disease symptoms. A positive medical response can be determined by measuring various objective parameters or criteria, for example, objective clinical signs of disease and / or improved survival. The medical response to the composition according to the invention can be easily verified in suitable animal disease models, which are well known in the art and are illustrated in the examples of this application.

[0118] The therapeutically effective amount will depend on the composition used, the route of administration, the species of mammal being treated (human or animal), the physical characteristics of the particular mammal under consideration, concurrent medications, and other factors recognized by those skilled in the medical arts.

[0119] In some embodiments, the pharmaceutical composition comprises another active agent, where the active agent is a pharmaceutical or therapeutic agent capable of preventing, treating or ameliorating disease in humans or animals. The active agent can be an antibody; an oligonucleotide, including antisense oligonucleotides, peptide nucleic acids (PNAs), small interfering RNAs, locked nucleic acids (LNAs), phosphorodiamidate morpholino oligonucleotides (PMOs), and decoy DNA molecules; a plasmid; an aptamer, including DNA, RNA, or peptide aptamers; a small or large chemical drug; or a protein, including a mixture thereof. In particular, the active agent can be an anti-cancer agent and / or an immunomodulatory agent. The anti-cancer agent can be a chemotherapeutic agent. The anti-cancer drug can also be another antibody, such as, but not limited to, Alacizumab, Amivantamab, Atezolizumab, BCD-100, Bemarituzumab, Bevacizumab, Cabiralizumab, Catumaxomab, Cetrelimab, Cetuximab, Ertumaxomab, Ficlatuzumab, Futuximab, Margetuximab, Necitumumab, Oportuzumab, Pancomab, Tomuzotuximab, and others. The immunomodulatory agent may be an anti-PD1 or anti-PDL1 agent, in particular an anti-PD1 or anti-PDL1 antibody; a cytokine, a mushroom glycan, a plant-derived immunomodulator, and an anti-cancer drug, a statin, a metformin, and an angiotensin receptor blocker (ARB), an anthracycline, a thalidomide, a lenalidomide, and a hypomethylating drug or others. The anti-cancer drug and / or the immunomodulatory agent may be advantageously bound to the antibody according to the invention by standard means known in the art, for example by covalent binding or by creating a genetic fusion.

[0120] The present invention also provides an antibody, a polynucleotide / vector or a pharmaceutical composition according to the present invention for use as a medicament.

[0121] The present invention also provides an antibody, peptide, polynucleotide / vector or pharmaceutical composition according to the present invention for use in the treatment of a disease involving the TRPV6 channel, particularly a disease associated with TRPV6 expression, such as cancer.

[0122] The present invention also provides an antibody, peptide, polynucleotide / vector, or pharmaceutical composition according to the present invention for treating a disease involving the TRPV6 channel, particularly a disease associated with TRPV6 expression, such as cancer.

[0123] The present invention also provides the use of the antibody, peptide, polynucleotide / vector according to the present invention in the manufacture of a medicament for the treatment of a disease involving the TRPV6 channel, particularly a disease associated with TRPV6 expression, such as cancer.

[0124] The present invention also provides pharmaceutical compositions for treating diseases involving the TRPV6 channel, particularly diseases associated with TRPV6 expression, such as cancer, which contain the antibodies, peptides, or polynucleotides / vectors according to the present invention as active ingredients.

[0125] The present invention also provides pharmaceutical compositions comprising the antibodies, peptides, and polynucleotides / vectors according to the present invention for treating diseases involving the TRPV6 channel, particularly diseases associated with TRPV6 expression, such as cancer.

[0126] As used herein, a disease associated with TRPV6 expression refers to a disease associated with altered TRPV6 expression, in particular, with high expression of TRPV6.

[0127] Diseases associated with TRPV6 expression may be selected from the group consisting of cancer; skin diseases, including but not limited to psoriasis, alopecia and dermatitis, epidermal proliferation disorders, skin aging, skin permeability, hyperphosphatemia and ectopic calcification; nervous system disorders, including but not limited to hearing loss and postpubertal goiter, preweaning, neuroexcitability, estrous cycle disorders and hypothalamic disorders, circadian rhythm, drug addiction, Parkinson's disease, pain sensation; gastrointestinal disorders, including but not limited to Crohn's disease, hypercalcemia, colonic crypt hypertrophy, irritable bowel syndrome; renal diseases, including but not limited to arterial and renal calcification, chronic kidney disease; bone mineral density and osteoporosis diseases and disorders; gynecological disorders, including but not limited to trophoblastic disorders, female infertility; and diabetes mellitus.

[0128] The cancer includes primary tumors and their metastases, and is preferably selected from the group consisting of endometrial cancer, leukemia, and carcinomas of the breast, pancreas, prostate, colon, ovary, and thyroid. In some further preferred embodiments, the cancer is prostate cancer.

[0129] The present invention also provides a method for treating a disease involving the TRPV6 channel, particularly a disease associated with TRPV6 expression, such as cancer, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition according to the present invention to a patient.

[0130] The pharmaceutical compositions of the present invention are generally administered in accordance with known procedures at dosages and for periods of time effective to induce a beneficial effect in an individual. Administration may be by injection or oral, sublingual, intranasal, rectal or intravaginal administration, administration by inhalation, or by transdermal application. Injection may be subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal or other injection.

[0131] The pharmaceutical compositions of the invention are advantageously used in combination with surgery, radiation therapy, chemotherapy and / or immunotherapy with immunomodulators. The combination therapy may be performed separately, simultaneously and / or sequentially.

[0132] In some embodiments, the pharmaceutical compositions are for use in treating human subjects.

[0133] In some embodiments, the compositions of the invention are used for the therapeutic treatment of individuals who have been previously diagnosed, e.g., using an antibody according to the invention, with a disease involving the TRPV6 channel, in particular a disease associated with TRPV6 expression.

[0134] Diagnostic and prognostic uses of antibodies Also an object of the present invention is the use of the antibody according to the present invention for the detection, diagnosis, prognosis and / or therapeutic outcome of diseases in which the TRPV6 channel is involved, in particular diseases associated with TRPV6 expression, such as cancer.

[0135] In this context, the present invention relates to the use of the antibody according to the invention in vitro in a biological sample from an individual as a diagnostic agent for the diagnosis of diseases in which the TRPV6 channel is involved, in particular diseases associated with TRPV6 expression, such as cancer.

[0136] In the present invention, (a) incubating an antibody according to the invention with a biological sample to produce a mixture; (b) detecting bound antibody in the mixture; The present invention provides an in vitro method in a biological sample obtained from an individual for detecting, diagnosing, predicting prognosis, and / or treating outcomes of diseases in which the TRPV6 channel is involved, particularly diseases associated with TRPV6 expression, such as cancer, comprising:

[0137] An antibody, preferably a labelled antibody as defined above, is used to detect expression of TRPV6 protein in an individual, which indicates that the individual has a disease, such as cancer.

[0138] For example, expression of TRPV6 protein can be detected in situ in tumor tissue from a patient compared to the same tissue from a healthy individual.

[0139] In some embodiments, step (b) comprises determining the amount of bound antibody in the mixture, and optionally comparing the amount of bound antibody in the mixture with at least one predetermined value.

[0140] In some embodiments, the method may include inferring from this value whether the individual is afflicted with a disease.

[0141] The present invention also relates to the in vitro use of the antibodies according to the invention for detecting the presence or determining the amount of TRPV6 protein present in a biological sample.

[0142] The present invention also provides a method for producing a semiconductor device comprising the steps of: a) contacting a biological sample with an antibody according to the invention; b) quantifying or detecting the presence or absence of bound antibody in the sample; c) estimating the amount or the presence or absence of TRPV6 protein in the sample from this value. The present invention relates to an in vitro method for detecting or determining the amount of TRPV6 protein in a biological sample, comprising:

[0143] The present invention also provides a method for producing a semiconductor device comprising the steps of: a) administering to an individual an antibody according to the invention, preferably a labeled antibody; b) detecting, quantifying, and / or localizing the antibody in the individual or a portion of the individual; The present invention relates to a method for diagnosing, prognosing, and / or treating a disease in an individual in which the TRPV6 channel is involved, in particular a disease associated with TRPV6 expression, such as cancer, comprising:

[0144] In some embodiments, the method may include inferring from this value whether the individual is afflicted with a disease.

[0145] The present invention also provides a method for producing a semiconductor device comprising the steps of: a) administering to an individual an antibody according to the invention; b) detecting, quantifying, and / or localizing the antibody in the individual or a portion of the individual; c) estimating from this value the presence or absence, amount and / or localization of TRPV6 protein in the individual or a part of the individual. The present invention relates to a method for detecting, quantitating or localizing TRPV6 protein in an individual or part of an individual, comprising:

[0146] When the antibody is administered to an individual, it is preferred to detect the antibody by in vivo imaging methods, in particular external methods such as two-dimensional or three-dimensional (3D) fluorescence imaging, or internal methods such as endoscopy.

[0147] The present invention also provides a method for producing a semiconductor device comprising the steps of: a) determining the level of TRPV6 protein in a biological sample using an antibody according to the invention; b) comparing the level of a) with a reference level of the protein, and determining that if the level of a) is higher than the reference level, the patient is suffering from an invasive disease (cancer) and has a poor prognosis. The present invention relates to a method for assessing the prognosis of a disease, such as cancer, associated with TRPV6 expression in a biological sample obtained from an individual, comprising:

[0148] Reference value refers to a value established by statistical analysis of values ​​obtained from a representative panel of individuals. This panel may depend, for example, on the nature of the sample, on the type of disease. Reference value can be obtained, for example, by measuring TRPV6 protein expression levels in a panel of normal individuals and / or individuals with non-invasive disease, for example non-invasive cancer, and determining a threshold value, for example a median concentration, which is used as reference value. When the method according to the invention is intended for patient monitoring, the reference value can be obtained from previously tested patients. High level refers to a significantly high level, i.e. a p-value of less than 0.1. Reference value is advantageously obtained from a panel of biological samples of the same type and / or from patients with the same type of disease, in particular the same type of cancer, as the patient being tested.

[0149] The above-mentioned prognosis prediction method may further comprise, after the comparison step, a further step c) of classifying patients into a good prognosis group and a bad prognosis group based on the TRPV6 level in the biological sample.

[0150] The above-mentioned prognosis prediction method can further comprise the further step of administering appropriate therapeutic drugs to each group of patients according to the severity of the disease after the step of classifying patients.The use of the prognosis prediction method of the present invention improves the treatment efficiency of diseases related to TRPV6 expression.

[0151] In particular, the patient is a newly diagnosed individual. Early assessment of the prognosis of cancer in the patient's primary tumor using the methods of the present invention allows the selection of the most efficient treatment for the patient: local radiotherapy for non-invasive tumors or systemic chemotherapy for invasive tumors.

[0152] In the above methods and uses, biological sample refers to biological material obtained from an individual that can be used for detection or diagnostic assay. The biological material can be derived from any biological source and is taken from a patient by standard methods well known to those skilled in the art. The biological sample is preferably a biopsy tumor cell or tissue, or a body fluid, such as serum, plasma, blood, lymphatic fluid, synovial fluid, pleural fluid, ascites or cerebrospinal fluid, mucus, bile, urine, saliva, tears, and sweat. In some embodiments, the biological sample is a biopsy tumor cell or tissue.

[0153] In the above method and use, TRPV6 protein expression can be quantified directly on the biological sample or after standard pretreatment according to the pretreatment method well known to those skilled in the art.Pretreatment can include, for example, cell lysis or embedding of biopsy tissue in plastic or paraffin.

[0154] In the above method and use, TRPV6 protein expression can be detected or quantified using various antibody-based techniques well known to those skilled in the art.Examples of such techniques include, but are not limited to, immunoassays, such as immunoblotting, immunoprecipitation, immunohistostaining, immunohistochemistry, immunofluorescence, such as flow cytometry, and FACS.Preferably, TRPV6 protein is detected or its level is measured using immunohistochemistry assays.Those skilled in the art will understand the parameters that may need to be manipulated to optimize the detection and / or quantification of TRPV6 protein using such techniques with anti-TRPV6 antibody according to the present invention.

[0155] Generated antibodies capable of detecting TRPV6 in the cell membrane are useful for any in vitro or in vivo detection or diagnostic immunoassay on living, fixed or denatured cells or tissues.

[0156] In some embodiments of the above methods and uses for the detection, diagnosis or prognosis of a disease, the disease is a cancer as defined above, including the primary tumor and its metastases. The cancer is preferably selected from the group consisting of endometrial cancer, leukemia, and carcinoma of the breast, pancreas, prostate, colon, ovary, and thyroid. In some further preferred embodiments, the cancer is prostate cancer.

[0157] In some embodiments of the above methods for detection, diagnosis or prognosis of disease, the patient is a human individual.

[0158] The above-mentioned methods and uses for the detection, diagnosis or prognosis of a disease according to the invention may be carried out simultaneously or subsequently on biological samples from different patients. The expression levels of other biomarkers may be measured in parallel.

[0159] Another subject of the present invention is a kit for the detection, diagnosis or prognosis of diseases in which the TRPV6 channel is involved, in particular diseases associated with TRPV6 expression, such as cancer, comprising at least an antibody according to the invention, preferably a labeled antibody, and optionally instructions for the use of the antibody.

[0160] The practice of the present invention will employ, unless otherwise indicated, conventional techniques within the skill of the art, such techniques being fully explained in the literature.

[0161] The present invention will now be illustrated by the following non-limiting examples and reference is made to the accompanying drawings in which: [Brief description of the drawings]

[0162] [Figure 1] Design and selection of epitope variants of different anti-TRPV6 channel antibodies raised against different peptide antigens. A. Schematic of the channel and relative location of epitopes bound by four polyclonal antibodies Ab79, Ab80, and Ab81 (basic image of channels from http: / / atlasgeneticsoncology.org / ). B. Schematic of the channel and relative location of epitope variant bound by monoclonal antibody 82 (basic image of channels from http: / / atlasgeneticsoncology.org / ). [Diagram 2] FIG. 1 is a schematic diagram showing the phage display panning strategy. [Diagram 3] Figure 5: Specificity ELISA of five IgGs. Evaluation of IgG binding at high concentrations to irrelevant peptide versus target peptide. [Figure 4]Figure 1 shows immunoblotting assays using anti-TRPV6 channel antibodies of the invention (Ab79 and 82) and commercially available antibodies. A. Shown are immunoblottings of whole cell lysates probed with various anti-TRPV6 antibodies, indicated on the bottom membrane surface, after LNCaP cells (TRPV6 positive) treated for 48 hours with either 40 nM siRNA-Luciferase (siCT) or siRNA-TRPV6 (siV6). B. Shown are immunoblottings using the same PVDF membrane as in BA, but probed with an anti-beta-actin antibody. [Diagram 5] Figure 1: Ab79a validation using knockdown / knockout and high expression models. A. Immunoblotting of LNCaP, HEK, CHO, and PNT1A cell lines expressing high TRPV6 channels using the vEF1ap-5'UTR-TRPV6_CMVp-mCherry vector (V6) compared to non-transfected cells. Total cell lysates were probed with Ab79a. B. Immunoblotting of PC3M, HAP-1wttrpv6+ / +, HAP-1trpv6- / -, and BSA protein compared to the housekeeping gene beta-actin (AKTB). [Figure 6] Flow cytometry diagram. White histograms represent secondary antibody alone, light grey histograms represent secondary antibody in addition to the antibody tested (Ab79, Ab82 or P3-R4-E11). [Figure 7]Figure 1 shows the effect of antibody treatment on calcium influx. A. Schematic of store-operated capacitative calcium entry (SOCE) in which TRPV6 channels play a key role. Inhibition of the SERCA pump by thapsigargin induces calcium leak, which releases calcium stores and activates store-operated channels (SOC), which in turn activates TRPV6 channels, which play a key role in amplifying calcium influx into the cell interior. B. Quantitative representation of SOCE in LNCaP cells prepared for 5 min with either glycerol (CT) or rabbit polyclonal anti-HA or polyclonal anti-TRPV6 antibody No:79 (79a). C. Quantitative representation of SOCE in LNCaP cells subjected to pretreatment with anti-TRPV6 antibody No:79 (79a) for 5 min and knocked down TRPV6 channels (siRNA, 40 nM, 48 h). D. Capacitative influx of calcium into the WT prostate cancer cell lines LNCaP (curve 1), LNCaP pretreated with Ab83 (curve 2), and LNCaP pretreated with Ab79 (curve 3). [Figure 8]Antibodies alter the current through the TRPV6 channel. A. Quantitative representation of TRPV6-specific currents from HEK cells transfected with vEF1ap-5'UTR-TRPV6wt_CMVp-mCherry vector and treated with rabbit polyclonal anti-TRPV6 antibody No:79 (79a) compared to control rabbit polyclonal anti-HA epitope antibody of the same isotype. n=3, *-p<0.05. B. Dose response curves with different dilutions of rabbit polyclonal anti-TRPV6 antibody No:79 (79a) above 0.5 μg / μl. C. Quantitative representation of TRPV6-specific currents from HEK cells transfected with vEF1ap-5'UTR-TRPV6wt_CMVp-mCherry vector and treated with mouse monoclonal anti-TRPV6 antibody No:82. Curves represent typical currents before and after stimulation of TRPV6 activity with DFV solution and after application of 1:5000, 1:2000, 1:1000, 1:500, and 1:200 dilutions of antibody as indicated. N equals: 1:5000 (n=9), 1:2000 (n=11), 1:1000 (n=13), 1:500 (n=25), and 1:200 (n=13). *-p<0.05. [Figure 9]FIG. 1 shows that TRPV6 modulation via polyclonal antibody 79 (79a) binding reduces cell survival. A. Cell survival assay (MTS) of LNCaP cells treated for 3 days with either anti-TRPV6 antibody No: 79 (79a) or control antibody anti-HA, with an equivalent amount of glycerol as control (CT). Dilutions are normalized to an initial amount of 0.5 μg / μl. n=3, *-p<0.05; **-p<0.01. B. Cell counts of LNCaP cells treated for 3 days with polyclonal anti-TRPV6 antibody No: 79 (79a) and also with antibody No: 80 and antibody No: 81 (raised against an intracellular epitope of the TRPV6 channel) and an isotype-matched anti-SERCA2B antibody, and glycerol (CT). n=3, **-p<0.01. C. Cell viability assay (MTS) of LNCaP cells treated for 3 days with either anti-TRPV6 antibody No:79 (79a) or a commercial anti-TRPV6 antibody (Alomone #ACC-036), with medium and an equivalent amount of glycerol as control (CT). Dilutions are normalized to an initial amount of 0.5 μg / μl. n=3, *-p<0.05; **-p<0.01. [Figure 10]Treatment of prostate cancer cells with polyclonal antibody 79 (79a) induces apoptosis in prostate cancer cells. A. Quantification of apoptosis rate assay using Hoechst staining of LNCaP cells. Cells were pretreated for 72 hours with either an equivalent amount of glycerol (CT) or polyclonal anti-TRPV6 antibody No: 79 (79a) (1 / 500, 0.5 μg / μl). Treatment with 1 μM thapsigargin (TG) for 72 hours was used as a positive control to induce apoptosis. n=3, **-p<0.01, ***-p<0.001. §-p<0.05 compared to treatment with TG (1 μM, 72 hours) alone. B. Quantification of apoptosis rate assay using Hoechst staining of HEK cells. n=3, **-p<0.01, ***-p<0.001. §-p<0.05 compared to treatment with TG (1 μM, 72 hours) alone. C. Quantification of sub-G1 peak of cell cycle assay of LNCaP cells treated for 72 hours with equivalent amounts of glycerol (CT) or with either polyclonal anti-TRPV6 antibody No:79 (79a) (1 / 500) or isotype-matched anti-HA. Treatment with 1 μM thapsigargin (TG) for 72 hours was used as a positive control to induce apoptosis. n=3, *-p<0.05; **-p<0.01. D. Trypan blue staining of LNCaP cells treated for 72 hours with equivalent amounts of glycerol (CT) or with polyclonal anti-TRPV6 antibody No:79 (79a) (1 / 500) or isotype-matched anti-HA and run for 8 hours, 24 hours, and 48 hours. n=3, *-p<0.05; **-p<0.01. [Figure 11] Figure 1 shows LNCaP prostate cancer cell survival. Cells were treated with either glycerol or control antibodies mabAU1 or mab82 for 1, 2, 3, and 4 days. n=3; *-p<0.05. [Figure 12]Figure 1 shows the effect of mouse monoclonal antibody mab82 (82a) on tumor growth and metastasis progression in vivo in immunodeficient mice. Mice were implanted with 2x10E6 cells derived from stable clones of PC3Mtrpv6- / --pmCherry and PC3Mtrp- / --pTRPV6wt cell lines. Mice were implanted in the dorsal neck and each group was divided into two groups for treatment with either control anti-AU1 or mab82 experimental antibody (82a) at the same dose of 100μg / kg. A. Growth of 1mm3 tumors measured every 3 days in control anti-AU1 or mab82 experimental antibody (82a) subgroups of PC3Mtrpv-+-+-pTRPV6wt group. Arrows indicate start of treatment. *-p<0.05; **-p<0.001. B. Survival curves of mice from both groups are shown. C. Growth of 1 mm3 tumors measured every 3 days in the control anti-AU1 or mab82 experimental antibody (82a) subgroups of the PC3Mtrpv6+ / +-mCherry group. Arrows indicate start of treatment. D. Growth of 1 mm3 tumors measured every 3 days in the PC3Mtrpv6+ / +-mCherry group vs. the PC3Mtrpv6- / --pTRPV6wt anti-AU1 subgroup. Arrows indicate start of treatment. *-p<0.05. E. Incidence of metastases in % between the PC3Mtrpv6- / --pmCherry and PC3Mtrpv6- / --pTRPV6wt groups and between both subgroups: control anti-AU1 or mab82 experimental antibody (82a). [Figure 13] Figure 1 shows the effect of antibody treatment on calcium influx.Capacitative calcium influx into WT prostate cancer cell line LNCaP pretreated with P3R4F03, P3R4E11, P3R5H03, and non-pretreated (CT) WT LNCaP. [Figure 14] Figure 1 shows the effect of antibody treatment on calcium influx.Capacitative calcium influx into humanized mab82, murine mab82, P2R4G08 pretreated WT prostate cancer cell line LNCaP, and non-pretreated (CT) WT LNCaP. [Figure 15]Figure 1 shows that TRPV6 modulation via P3R4F03 antibody binding reduces cell survival. Cell viability assay (Cell titer glo) of LNCaP cells treated for 3 days with either an equivalent amount of irrelevant human IgG1 antibody as control (IA) or anti-P3R4F03 antibody. Dilutions are normalized to an initial amount of 0.5 μg / μl. n=3, * p<0.05; ** p<0.01; *** p<0,001. EXAMPLES

[0163] Materials and Methods Peptide epitopes Peptide epitopes (peptide antigens) are derived from the human TRPV6 sequence UniProtKB / Swiss-Prot: NP_061116.5 or Q9H1D0.3 (SEQ ID NO: 1). Peptides were synthesized and purified to greater than 99% purity.

[0164] Generation of polyclonal antibodies Peptide epitopes (peptide antigens) were conjugated to the N-terminus of KLH protein and injected into rabbits once a week for 4 weeks before terminal bleeding (Eurogentec, LTD). Sera were tested by ELISA using antigen-coated plates and then affinity purified against the same bound antigen in a column. The final affinity-purified antibodies were supplied, diluted 50 / 50 v / v in glycerol and stored at -20°C.

[0165] Generation, cloning, and characterization of monoclonal antibodies The manufacturing process was standard and consisted of four consecutive immunizations with the same peptide antigen. One week after the fourth boost, serum samples were tested and suitable antibody-bearing animals were selected, sacrificed and their B-lymphocytes were fused with hybridomas. Once sufficient titers were obtained, mAb samples were tested again to select the most efficient / promising samples. Hybridomas were then expanded and antibodies were isolated and affinity purified on a column against the epitope peptide.

[0166] Primers and siRNA

[0167] [Table 1]

[0168] reagent All reagents were purchased from Sigma (Sigma, L'Isle d'Abeau Chesnes, France) unless otherwise specified.

[0169] cell culture Human PC3M (a metastatic cell line derived from PC3 cells engrafted in vivo), PC-3M, LNCaP, PNTA1, HEK293, and CHO-K1 cell lines were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI (LNCaP, PC3M, PNT1A), DMEM (HEK293), and F12 (CHO) medium (Gibco-BRL) supplemented with 10% fetal bovine serum and containing kanamycin (100 μg / ml) and L-glutamine (2 mM) as required. The HAP1 cell line is a near-haploid human cell line, derived from the male chronic myeloid leukemia (CML) cell line KBM-7 (Carette et al., Nature. 2011, 477, 7364, 340-3) and cultured in IMDM medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum and containing kanamycin (100 μg / ml) and L-glutamine (2 mM). The PNT1a cell line was obtained from the American Type Culture Collection (ATCC) and cultured in RPMI. All cells were cultured in a humidified atmosphere at 37°C with 5% CO2 in air. The medium was changed three times a week, and cultures reached confluence when cells were detached by treating them with 0.25% trypsin (in PBS) for 5 min at 37°C. For this experiment, cells were seeded in 6-well plates for PCR and Western blotting. Cellular trpv6 - / -To maintain the condition, the selected antimicrobial agent G418 was administered at a concentration of 200 μg / ml to HAP1. trpv6- / - For the maintenance of cell culture, PC3M and puromycin were added at a concentration of 0.1 μg / ml. trpv6- / - It was used for maintaining the cell line culture.

[0170] For antibody treatment, serum was either thoroughly withheld, ie heated at 62° C. for 1 hour under constant agitation, or in some cases serum-free medium, such as AIM V from Gibco™, was used.

[0171] Electrophysiology and Solutions Currents from HEK-293 cells transfected with the vEF1ap-5'UTR-TRPV6_CMVp-mCherry vector were recorded macroscopically in the whole-cell configuration of the patch clamp technique using a computer-controlled EPC-9 amplifier (HEKA Electronic, Germany) as described previously (Raphael et al., 2014). The composition of the extracellular solution for patch clamp recordings was 120 mM NaCl, 5 mM KCl, 10 mM CaCl2, 2 mM MgCl2, 5 mM glucose, 10 mM HEPES, pH adjusted to 7.4 with TEA-OH and osmolality adjusted to 310 mOsm / kg with D-mannitol. Patch pipettes were filled with intracellular pipette base solution: 120 mM Cs-methanesulfonate, 10 mM CsCl, 10 mM HEPES, 10 mM BAPTA (1.2-bis(2-aminophenoxy)ethane N,N,N',N'tetraacetic acid), 6 mM MgCl2 (adjusted to pH 7.4 with CsOH and osmolarity 295 mOsm / kg with D-mannitol). Required supplements at the desired concentrations were added directly to the experimental solutions from the appropriate stock solutions, dissolved in water, ethanol or dimethyl sulfoxide. All chemicals were purchased from Sigma-Aldrich. During patch clamp recordings, drugs and solutions were applied to the cells by placing a multi-line microperfusion system with a common outflow (Cell Micro Controls, Norfolk, VA) in close proximity (approximately 200 μm) to the test cells. Experiments were performed at room temperature.

[0172] Calcium Imaging Cells were seeded on cover slips and 4 μM Fura-2 AM was added to the growth medium for 45 min at room temperature. Recordings were performed in HBSS containing 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 0.3 mM Na2HPO3, 0.4 mM KH2PO4, 4 mM NaHCO3, 5 mM glucose, and 10 mM HEPES, NaOH adjusted to pH 7.4. CaCl2 was adjusted to 0.07 mM or 1.8 mM depending on the experiment. The cover slip was then placed on the microscope stage in a perfusion chamber. Fluorescence images of the cells were recorded by a video image analysis system (Quanticell). Fura-2 fluorescence at emission wavelengths of 510 nm or alternatively at 340 nm and 380 nm was recorded by exciting the probe. The 340 / 380 nm signal ratio was calculated using an in vitro calibration to determine [Ca 2+ ] i converted to levels.

[0173] SDS-PAGE and Western blotting Semi-confluent cells were treated with ice-cold lysis buffer containing 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM MgCl, 1 mM PMSF, 1% Nonidet P-40, and Sigma protease inhibitor cocktail. The lysates were centrifuged at 15,000 × g for 20 min at 4 °C, mixed with sample buffer containing 125 mM Tris-HCl pH 6.8, 4% SDS, 5% β-mercaptoethanol, 20% glycerol, 0.01% bromphenol blue, and boiled for 5 min at 95 °C. Total protein samples were subjected to 8%, 10%, and 15% SDS-PAGE and transferred to nitrocellulose membranes by semi-dry Western blotting (Bio-Rad Laboratories). The membrane was blocked overnight in 5% milk containing TNT buffer (Tris-HCl pH 7.5, 140 mM NaCl, and 0.05% Tween 20) and then probed with a specific rabbit polyclonal anti-TRPV6 antibody (all diluted 1 / 500) and a mouse monoclonal anti-β-actin antibody (Lab Vision Co., 1 / 1000). Bands on the membrane were visualized using enhanced chemiluminescence (Pierce Biotechnologies Inc.). Densitometric analysis was performed using a Bio-Rad image acquisition system (Bio-Rad Laboratories).

[0174] Flow cytometry 200,000 cells were incubated with 15 μg / mL of polyclonal rabbit antibody pAb79 or monoclonal mouse antibody mAb82 for 1 h on ice. IgG binding was detected using 10 μg / mL of anti-rabbit AF488 or anti-mouse AF488 antibodies (A-11034 and A-11029, respectively, from Invitrogen) or P3-R4-E11 for 1 h on ice. IgG binding was detected using anti-Fab-AF647 (Jackson-109-605-006). Analysis was performed by flow cytometry.

[0175] RT-PCR RT-PCR experiments were performed as previously described (Lehen'kyi et al., 2007). Total RNA was isolated using a guanidinium thiocyanate-phenol-chloroform extraction procedure. After removal of genomic DNA by DNase I (Life Technologies), 2 μg of total RNA was reverse transcribed into cDNA at 42°C using random hexamer primers (Perkin Elmer) and MuLV reverse transcriptase (Perkin Elmer) in a final volume of 20 μl, followed by PCR as described below. The PCR primers used for amplification of TRPV6 cDNA as well as primers for AR, VDR, and β-actin are specified in Table XVI above. PCR was performed on the RT-generated cDNA using a GeneAmp PCR System 2400 thermal cycler (Perkin Elmer). To detect the various cDNAs, PCR was performed by adding 1 μl of RT template to a mixture (final concentrations) of 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgCl2, 200 μM of each dNTP, 600 nM sense and antisense primers, and 1 U of AmpliTaq Gold (Perkin Elmer) in a final volume of 25 μl. The DNA amplification conditions were an initial denaturation step of 95° C. for 7 min, followed by 40 cycles of 95° C. for 30 s, 60° C. for 30 s, 72° C. for 30 s, and a final 72° C. for 7 min. The primers used were those listed in the table above.

[0176] Quantitative real-time PCR Quantitative real-time PCR of TRPV6 and HPRT mRNA transcripts was performed on Biorad's CFX96 real-time PCR detection system using MESA GREEN qPCR MasterMix Plus for SYBR Assay (Eurogentec). Primer sequences are shown in Table XVI. The HPRT gene was used as an endogenous control to normalize for variations in RNA extraction, RNA degradation, and RT efficiency variability. To quantitate the results, the comparative threshold cycle method ΔΔCt and Biorad's CFX Manager Software v2.0 were used.

[0177] siRNA transfection HAP1 cells were transfected with 40 nM siRNA against TRPV6 (1-4 or mixture) or siLuciferase (Eurogentec, LTD, Belgium) using 5 μl of Lipofectamine 3000 transfection reagent (Thermofisher) according to the manufacturer's instructions (see Table I for siRNA sequences). Cell transfection efficiency with siRNA against each specific target was verified using real-time quantitative PCR and / or Western blotting, as appropriate.

[0178] Nucleofection Transfection of the different cell lines with the different plasmids was performed using a Nucleofector (Amaxa GmbH) according to the manufacturer's instructions. Briefly, 2 μg of plasmid was transfected into 2 million trypsinized cells, which were then seeded onto 6-well dishes, 35 mm dishes or cover slips for 48 h.

[0179] Cell viability assay Cell proliferation was measured using the CellTiter 96 Aqueous One Solution cell proliferation assay (Promega) based on the intracellular conversion of the colorimetric reagent MTS [3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt] to soluble formazan by dehydrogenase enzymes found only in metabolically active proliferating cells. After each treatment, 20 μl of the dye solution was added to each well in a 96-well plate and incubated for 2 hours. The absorbance at a wavelength of 490 nm was then recorded using an ELISA plate reader (Molecular Devices). The percentage of cell proliferation inhibition was calculated as (A 対照 -A 試料 ) / (A 対照-A ブランク ) x 100%.

[0180] To evaluate P3R4F03, the CellTiter-Glo® Luminescent Cell Viability Assay was used to measure cell viability based on the firefly luciferase reaction, which converts luciferin to light, to ATP production by metabolically active proliferating cells. Cells were treated with equal doses of either an irrelevant antibody (IA) or P3R4F03. After each treatment, 100 μl of CellTiter-Glo® Reagent solution was added to each well in a 96-well plate and incubated for 10 min. Luminescence was then recorded using an ELISA plate reader (Polar Star Omega, BMG Labtech, Germany). Percentage of cell proliferation inhibition was calculated as (A 対照 -A 試料 ) / (A 対照 -A ブランク ) x 100%.

[0181] Cell cycle assay Flow cytometry assays were performed on triplicate 25 cm 2 This was performed on a cell population cultured in a flask. 6Cells were fixed with 1 ml of ice-cold 70% methanol for 30 min. After fixation, cells were pelleted by centrifugation to remove the fixative, washed three times with phosphate-buffered saline (PBS) at 4°C, resuspended in 100 μl PBS, treated with 100 μl RNAse A (1 mg / ml, Sigma), and stained with propidium iodide (PI, Sigma) at a final concentration of 50 μg / ml. Stained cells were stored in the dark at 4°C and analyzed within 2 h. Stained samples were measured on a FACScan flow cytometer (Becton-Dickinson, San Jose, CA). Data were acquired with a coefficient of variation of less than 5% for 7000 events, and red fluorescence was measured on the X-axis using a fluorescence detector 3 (FL3). Data were stored and analyzed using CellQuest software to evaluate cell cycle distribution patterns (subG1 (apoptotic), G0 / G1, S, and G2 / M phases).

[0182] TUNEL assay The level of apoptosis was estimated from the number of apoptotic nuclei revealed by either TUNEL-TMR Red assay (Roche Biochemicals) or Hoechst staining. The percentage of apoptotic cells was determined by counting at least five random fields for each condition performed in triplicate for each "n".

[0183] Immunohistochemistry Paraffin-processed anonymous human prostate tissue sections from eight prostatectomies were obtained from the Department of Cytopathology, Hôpital St Vincent de Lille, Lille. Tumors were fixed immediately after resection and paraffinized according to conventional procedures, then cut at 7 μm on a microtome and stacked on slides. Paraffin-embedded prostate sections were subjected to conventional deparaffinization, followed by antigen retrieval using citrate buffer at 95°C in a water bath. After saturation with a solution containing 1% BSA and 0.05% Triton X100 in PBS-gelatin, prostate sections were incubated overnight at 4°C with specific antibodies, e.g. rabbit polyclonal anti-TRPV6 antibodies (No: 79a-c, 80-82, 1 / 200). Donkey or goat polyclonal anti-rabbit peroxidase-conjugated secondary antibodies and donkey or goat polyclonal anti-mouse peroxidase-conjugated secondary antibodies (Chemicon International, 1 / 200) were used. After development with diaminobenzidine (Sigma-Aldrich), slides were mounted with Glycergel® and images were analyzed using a Zeiss Axioskope microscope (Carl Zeiss) and Leica Image Manager software (Leica Geosystems AG). Immunohistochemistry was performed automatically using a Benchmark XT automated slide stainer (Ventana Medical Systems, Inc., Tucson, AZ) according to established protocols, and detection was performed using the IVIEW-DAB detection system (N760-500, Ventana Medical Systems, Inc.).

[0184] Plasmids The entire TRPV6 cDNA including the 5'-UTR was used on pCAGGS to obtain the final vEF1ap-5'UTR-TRPV6_CMVp-mCherry vector (E-Zyvec, France), which was nucleofected into cells and the transfection rate was evaluated using the control vEF1ap-5'UTR_CMVp-mCherry vector. The pTRPV6-eYFP and pOrai1-YFP vectors were used as previously described (Raphael et al., 2014).

[0185] Animals, antibody injections, tumorigenicity assays, and surgery Studies on animals, including housing and care, euthanasia procedures, and experimental protocols were performed in accordance with the Animal Ethics Committee (approval number 201703021400830) in the animal house of the University of Lille (Cite Scientifique campus) (permit: C59-00913) and under the supervision of Dr. Lehen'kyi (permit: 59-009270). Tumor cells (2 × 10 6 Cells / mouse) were injected subcutaneously into 6-week-old male Swiss nude mice (Charles-Rivers, France) with 50% (v:v) Matrigel (BD biosciences). For antibody studies, mice were randomized for treatment (at least 10 animals / group) once tumors started to become visible and were administered intraperitoneally twice a week with either anti-AU1 or anti-TRPV6 mab82 antibodies at 100 μg / kg diluted in PBS. Mice were sacrificed if animal welfare was compromised. When tumors reached their maximum recognized size, animals were submitted to surgery, i.e., tumors were excised. Tumors were dissected, photographed, weighed, and volume determined. For metastasis studies, animals were monitored daily and mCherry imaging was performed using a small animal imaging system (Bruker, USA).

[0186] scFv screening A three-phage display selection strategy was performed on TRPV6 peptides and PC3M luc C6 cells using the iMAb Corporation HuscIII proprietary library (Figure 18). Depletion was performed on streptavidin as described in Figure 4. Four first selection rounds on peptides and one fifth selection round on cells were performed using the depleted library.

[0187] In the first two rounds, biotinylated TRPV6 peptides were immobilized on streptavidin-coated maxisorp plates. Phages from each selection round (10 10 phage / mL) and detected using anti-M13 antibody-HRP.

[0188] For scFvs selected from the first two rounds, expression was induced, culture supernatants containing secreted scFvs were harvested, and scFv binding to TRPV6 peptide was assessed by ELISA for the third, fourth, and fifth rounds.

[0189] In the third, fourth, and fifth rounds, biotinylated peptides were immobilized on streptavidin-coated maxisorp plates. ScFv products (culture supernatants) were added and detected using anti-c-myc-HRP.

[0190] SDS-PAGE analysis of purified IgG Proteins were either reduced or not, and 1.6 μg of protein was loaded onto each lane on a 4-20% gel.

[0191] Dose response ELISA TRPV6 biotinylated peptide was immobilized on streptavidin-coated plates at 10 μg / mL. IgG binding was tested at various concentrations (0.00282 nM to 500 nM) and detected using anti-Fab-HRP (Sigma A0293).

[0192] specific ELISA TRPV6 and an unrelated biotinylated peptide were immobilized on streptavidin-coated plates. IgG binding was examined at high concentrations (75 μg / mL to 500 nM) and detected using anti-Fab-HRP (Sigma A0293).

[0193] 4. Data Analysis For each experiment, data from at least three measurements were accumulated. Data were analyzed using Origin 7.0 software (Microcal Software Inc., Northampton, MA). Results were expressed as mean ± SEM, where appropriate. N equals the number of experiments in a series, and n equals the number of cells used in the study. ANOVA was used for statistical comparison of differences, and P<0.05 was considered significant. In the graphs, (*) and (**) indicate statistically significant differences with P<0.05 and P<0.01, respectively.

[0194] Example 1 Design and validation of anti-TRPV6 antibodies raised against extracellular epitopes Selection of epitopes for rabbit polyclonal antibody No. 79. Thirty-seven amino acids span the first extracellular loop, located between the S1 and S2 transmembrane domains. Three of these residues are asparagine, N residues. Detailed analysis using NetNGlyc 1.0 software demonstrated the most likely secondary and tertiary sites of N-glycosylation, RTNNRT, and RDNTL. The presence of such sites and these N-glycosylations negates the possibility of steric contacts by the antibody to the epitope. On the other hand, the lipid bilayer prevents the antibody from binding to the respective amino acids of the epitope. The three epitopes were used to generate rabbit polyclonal antibodies, designated 79a-c. These three epitopes correspond to peptide 79a or hTRPV6 414-428; hTRPV6 sequence, 414-428 bases; QEAYMTPKDDIRLVG (SEQ ID NO: 3), peptide 79b or hTRPV6 414-425; hTRPV6 sequence, 414-425 bases; QEAYMTPKDDIR (SEQ ID NO: 4), and peptide 79c or hTRPV6 412-425; hTRPV6 sequence, 412-425 bases; LLQEAYMTPKDDIR (SEQ ID NO: 5), respectively. Their efficiency in recovering antigens was demonstrated using a series of immunoblotting assays under denaturing conditions.

[0195] Monoclonal Antibody 83 One monoclonal antibody was raised against the peptide EAYMTPKEEIRR (SEQ ID NO: 7), which is a variant of the peptide hTRPV6 415-426 (EAYMTPKDDIRL; SEQ ID NO: 8), located at the C-terminus of the X-loop between the S1 and S2 transmembrane domains.

[0196] Selection of epitopes for monoclonal antibody 82 One monoclonal antibody was raised against a peptide located at the N-terminus of the p-loop (between S5 and S6) in the pore region (Figure 1). Different antigenic peptides (peptide epitopes) located in the target sequence hPRV6 551-573 (IFQTEDPEELGHFYDYPMALFST; SEQ ID NO: 9) were tested: peptide 82a (hPRV6 553-570; QTEDPEELGHFYDYPMAL; SEQ ID NO: 10); peptide 82b (hPRV6 551-567; IFQTEDPEELGHFYDYP; SEQ ID NO: 11); peptide 82c (hPRV6 557-573; PEELGHFYDYPMALFST; SEQ ID NO: 12); peptide 82d (hPRV6 554-568; TEDPEELGHFYDYPM; SEQ ID NO: 13); peptide 82 (hPRV6 554-569; TEDPEELGHFYDYPMA; SEQ ID NO: 14). Monoclonal antibody 82 (mab82), raised against peptide 82 (hPRV6 554-569), was further characterized.

[0197] Design and validation of anti-TRPV6 antibodies generated against extracellular epitopes by phage display Three peptides of human TRPV6 were synthesized by Genosphere Biotechnologies, Inc. One was biotinylated at the N-terminus and the other two at the C-terminus. Their main properties are summarized in Table III.

[0198] [Table 2]

[0199] Peptide 1 and peptide 2 were previously used for peptide immunization to discover pAb79 and mAb82, respectively, while peptide 3 is an additional peptide designed to target another loop in the pore-forming region.

[0200] Three phage display selection strategies were performed on TRPV6 peptides and PC3M luc C6 cells using the iMAb HuscIII proprietary library (Figure 2). Depletion was performed on streptavidin as described in Figure 2. Four first selection rounds on peptides and one fifth selection round on cells were performed using the depleted library. After the first three selection rounds, the results of the three panning strategies were tested for binding to TRPV6 peptides by polyclonal ELISA using the phage pools selected in each panning round and anti-M13-HRP as secondary antibody. Enrichment of TRPV6 peptide binders was observed in all selections. After the first three selection rounds, 93 individual colonies (containing a single scFv) from each of the three selections were picked and expanded. scFv expression was induced and culture supernatants containing secreted scFvs were harvested to evaluate scFv binding to TRPV6 peptides by ELISA. Anti-c-myc-HRP antibody was used to detect scFvs. In summary, 21 clones were found to be positive at the end of the third round: 3 for peptide 1, 7 for peptide 2, and 11 for peptide 3. To further enrich the selection of TRPV6 binders, it was decided to perform two additional rounds for peptides and cells, respectively. The results of the fourth and fifth round selections were screened as previously described.

[0201] In summary, 22 clones were found to be positive at the end of the fourth round: 6 for peptide 1, 7 for peptide 2, and 9 for peptide 3.

[0202] In summary, four clones: two for peptide 1 and two for peptide 3 were found to be positive at the end of the fifth round.

[0203] After the third, fourth, and fifth screening rounds, 47 scFvs were shown to specifically bind to the TRPV6 peptide.

[0204] 47 scFvs were selected by ELISA screening and sent for sequencing. Thirty-four of these were unique sequences: 8 for peptide 1, 11 for peptide 2, and 15 for peptide 3. Sequences with high sequence identity were grouped into clusters. Further ELISAs were performed to confirm specific binding of unique clones. ScFv expression was induced, culture supernatants were harvested, and scFv binding to TRPV6 peptide and an unrelated peptide was evaluated in triplicate. ScFvs were detected using anti-c-myc-HRP antibody. Eleven scFvs were selected for further characterization in IgG format, and the selected eleven scFvs were subcloned into human IgG1 format expressed at small scale in HEK293T cells and purified on protein A beads. The SDS-PAGE migration profile of nine of the eleven was comparable to that of the control IgG (trastuzumab). Two IgGs showed high molecular weights, corresponding to N-glycosylation. This was confirmed by sequence analysis (data not shown).

[0205] IgG binding to human TRPV6 peptide was examined by ELISA. TRPV6 biotinylated peptide was immobilized on streptavidin-coated plates at 10 μg / mL. IgG binding was examined at various concentrations (0.00282 nM to 500 nM) and detected using anti-Fab-HRP (Sigma A0293). IgGs P2-R4-G8, P3-R4-F3, P3-R4-E11, P3-R5-E6, and P3-R5-H3 showed strong binding to TRPV6 peptide, allowing the determination of EC50 (Table IV).

[0206] [Table 3]

[0207] The specificity of the IgG was then evaluated by ELISA. TRPV6 and an irrelevant biotinylated peptide were immobilized on streptavidin-coated plates. IgG binding was tested at high concentrations (75 μg / mL to 500 nM) and detected using anti-Fab-HRP (Sigma A0293). No binding to the irrelevant peptide was observed for IgGs P2-R4-G8, P3-R4-F3, P3-R4-E11, P3-R5-E6, and P3-R5-H3, indicating high specificity of the IgG for the selected peptides (Figure 3).

[0208] Detection of TRPV6 peptide expression using various rabbit polyclonal antibodies Three polyclonal antibodies (Ab79a, Ab79b, Ab79c) were raised against a peptide located at the C-terminus of the X-loop between the S1 and S2 transmembrane domains (Figure 1). Antibody reactivity was quantified by immunoblotting of various total cell lysates, LNCaP (Figures 4 and 5A) and PC-3M (Figure 5B) (i.e., TRPV6 positive). Bands of approximately 95-100 kDa, the size expected for the glycosylated / mature form of the TRPV6 channel, were observed in LNCaP cells for ab79a, ab79b and ab79c (Figure 4A). A non-specific 50 kDa band was also detected by Ab79a-c (Figure 4B). Numerous small sized bands were observed, mostly with Ab79c (Fig. 4A), and no reliable staining was observed with commercially available anti-TRPV6 antibodies (Sigma SAB2106366 and Santa-Cruz sc-28763) designed to recognize intracellular epitopes of human TRPV6 (Fig. 4A).

[0209] As a result, immunoblotting under denaturing conditions allowed the assessment of antibody specificity for the particular epitope to which the antibody was raised. Ab79a was the better antibody, able to detect a band of the expected size for monomeric TRPV6 protein.

[0210] Detection of TRPV6 protein expression using mab82 antibody One monoclonal antibody was raised against a peptide located at the N-terminus of the p-loop (between S5 and S6) in the pore region (Figure 1). Antibody reactivity was quantified by immunoblotting of various whole cell lysates, LNCaP (Figure 4B) (i.e., TRPV6 positive). A band of approximately 95-100 kDa, the expected size for the glycosylated / mature form of the TRPV6 channel, was observed in LNCaP cells only with mab82; no reliable staining was observed with commercially available anti-TRPV6 antibodies (Sigma SAB2106366 and Santa-Cruz sc-28763) designed to recognize an intracellular epitope of human TRPV6.

[0211] As a result, immunoblotting under denaturing conditions allowed the assessment of antibody specificity for the particular epitope to which the antibody was raised. mAb82 was the only antibody capable of detecting a band of the expected size for monomeric TRPV6 protein (compared to the commercial antibodies tested).

[0212] Validation of the 79a antibody using knockdown, (high)expression, and knockout models In this study, four siRNAs were used to perform specific knockdown of TRPV6 expression. The list of siRNA sequences is shown in Table I (Table 4). They target the first, seventh, eleventh, and thirteenth exons of the mRNA. First, quantitative real-time PCR of TRPV6 channel was performed relative to HPRT gene expression in LNCaP cells transfected with 40 μM of control siRNA (luciferase) or 40 μM of either siRNA 1-4 against TRPV6 channel or their mixture. The knockdown of mRNA attenuation level was more than 60% efficient, as reflected by the corresponding immunoblotting and quantification of bands of protein lysates from siRNA-treated LNCaP cells compared to AKTB (data not shown).

[0213] As a next step, a (high) expression system was used. It should be noted that it is extremely difficult to have an in vitro cell line that does not express the TRPV6 channel, since the presence of 2 mM calcium in almost all media favors TRPV6 expression for cell survival. The data showed that TRPV6 expression ranged from a slight increase to a strong increase, suggesting that the band at approximately 100 kDa was specific.

[0214] Flow cytometry To test the specificity of the antibody against TRPV6 PC3M expressing TRPV6, we tested luc C6 transfected cell lines and PC3M KO cell lines. The results showed that Ac 79, 82, or P3-R4-E11 showed strong binding to PC3M luc C6 cells, but a weak signal was observed for PC3M KO (Figure 6).

[0215] Example 2 Use of anti-TRPV6 antibodies raised against extracellular epitopes for cancer diagnosis and prognosis in clinical samples Immunohistochemistry (IHC) was performed using rabbit polyclonal anti-TRPV6 antibody No. 79a using human clinical samples from prostatectomy specimens, including normal prostate, bladder cancer resection specimens, and adenocarcinoma with Gleason score 7 (data not shown).

[0216] These data confirm the negative expression of TRPV6 channel in healthy prostate, which is consistent with previous published data (Wissenbach et al. 2001; 2004; Peng et al. 2001; Raphael et al. 2014). Therefore, rabbit polyclonal anti-TRPV6 antibody No. 79a can be used for diagnostic / prognostic purposes. Finally, HAP-1 trpv6- / - Cell lines and HAP-1 trpv6+ / + IHC of tumor slices derived from tumors implanted using the cell line was performed using rabbit polyclonal anti-TRPV6 antibody No. 79a. The results showed that rabbit polyclonal anti-TRPV6 antibody No. 79a inhibited HAP-1trpv6- / - We show that no TRPV6 channels are recognizable in the forming tumors, validating both the knockout model and the antibody specificity.

[0217] Immunohistochemistry (IHC) was performed using mouse monoclonal mab82a antibody using prostatectomy specimens containing normal prostate (bladder cancer resection specimens) and human clinical samples from adenocarcinoma with Gleason score 7.

[0218] These data confirm the negative expression of TRPV6 channels in healthy prostate, which is consistent with previous published data (Wissenbach et al. 2001; 2004; Peng et al. 2001; Raphael et al. 2014). Therefore, the rabbit polyclonal anti-TRPV6 antibody mab82a can be used for diagnostic / prognostic purposes.

[0219] Example 3 Treatment with anti-TRPV6 antibodies raised against extracellular epitopes modulates TRPV6 channel activity Antibody treatment increases store-operated capacitative calcium entry in PCa cells TRPV6 has been shown to be a key component of store-operated calcium entry (SOCE) into PCa cells, allowing the use of this mechanism to detect and analyze TRPV6 activity (Raphael et al., 2014). This mechanism is triggered by the release of calcium stores in the endoplasmic reticulum (ER). Inhibition of the SERCA pump with thapsigargin (1 μM) is used to induce calcium leak. This releases calcium stores, activating store-operated channels (SOCs), such as Orai1 or TRPC1, which then activates the TRPV6 channel, which plays at least a half-important role in amplifying calcium entry into the cell interior (Raphael et al., 2014; Figure 7A).

[0220] In the experimental protocol, cells are first incubated with a calcium-free solution to generate an outward gradient, which is amplified by the use of 1 μM thapsigargin, which blocks the SERCA pump, thereby disabling calcium reuptake into the ER. This artificial condition results in a significant lack of calcium, which is important for cell survival, and opens the so-called store-operated channels (SOC). Addition of 2 mM calcium results in calcium influx through SOC, which then activates the TRPV6 channel, which plays a key role in amplifying calcium influx into the cell interior (Raphael et al., 2014). Pre-incubation of PCa cells, e.g., LNCaP, for 5 min with either glycerol (CT) or rabbit polyclonal anti-HA or polyclonal anti-TRPV6 antibody No. 79a, all normalized to 0.5 μg / μl with a 1 / 500 dilution, caused differential effects, e.g., a selective and significant increase in SOCE levels in the case of polyclonal antibody No. 79a (Figure 7B). To verify that such effects are mediated by the TRPV6 channel, we controlled the antibodies during the use of the siRNA strategy for TRPV6 knockdown. SOCE was significantly reduced during TRPV6 channel knockdown, and the increase in SOCE mediated by antibody No. 79a (siCT+No. 79a) was significantly attenuated compared with siTRPV6+No. 79 treatment (Figure 7C). Thus, both polyclonal antibodies of No. 79 activate TRPV6, which amplifies SOCE, and further allows large amounts of calcium to enter the cell.

[0221] Preincubation of cells with antibody 83 resulted in an increase in capacitative calcium influx above controls, indicating that antibody 83, like antibody 79, increases TRPV6-mediated calcium influx (Figure 7D).

[0222] Polyclonal antibody 79a directly affects TRPV6-induced currents The gold standard for extracellular antibody action against ion channels is the electrophysiological technique, which allows the measurement of ionic currents passing through specific channels, since each of these antibodies has unique conductive features or properties. The specificity of the developed polyclonal antibody No. 79 was verified by measuring its effect on whole-cell currents recorded from HEK cells transfected with vEF1ap-5'UTR-TRPV6wt_CMVp-mCherry (Figure 8). First, 10 mM Ca, known to block TRPV6 activity, was used as described. 2+ The cells were bathed in a physiological solution containing HA (Singh et al., Sci Adv. 2018, 4, eaau6088; Derler et al., J Physiol. 2006, 577, 31-44; Niemeyer et al., Proc Natl AcadSci US A. 2001, 98, 3600-5). TRPV6-specific currents were induced by replacing the extracellular (bath) solution with a divalent cation-free (DVF) solution, which is generally known to stimulate TRPV6 activity (Derler et al., 2006; Niemeyer et al., 2001). The rabbit polyclonal anti-TRPV6 antibody No. 79a was able to significantly increase the current while binding to the TRPV6 channel compared to the control antibody, a rabbit polyclonal anti-HA epitope antibody of the same isotype (1 / 500, 0.5 μg / μl) (Figure 8A). To confirm the specificity of this binding, dose-response experiments were performed, which showed a progressive activation of TRPV6 channels (FIG. 8B).

[0223] Monoclonal antibody 82 directly affects TRPV6-induced currents The gold standard for extracellular antibody action against ion channels is the electrophysiological technique, which allows the measurement of ionic currents passing through specific channels, since each of these antibodies has unique conductance characteristics or properties. The specificity of monoclonal mab82 was verified by measuring its effect on whole-cell currents recorded from HEK cells transfected with vEF1ap-5'UTR-TRPV6wt_CMVp-mCherry (Figure 8C). First, as described, 10 mM Ca, known to block TRPV6 activity, was used. 2+ The cells were bathed in a physiological solution containing TRPV6 (Singh et al., Sci Adv. 2018, 4, eaau6088; Derler et al., J Physiol. 2006, 577, 31-44; Niemeyer et al., Proc Natl AcadSci US A. 2001, 98, 3600-5). TRPV6-specific currents were induced by replacing the extracellular (bath) solution with a divalent cation-free (DVF) solution, which is generally known to stimulate TRPV6 activity (Derler et al., 2006; Niemeyer et al., 2001). The mouse monoclonal anti-TRPV6 antibody mab82a was able to significantly reduce the current while binding to the TRPV6 channel compared to the control antibody (CT) (Figure 8C). The specificity of the developed monoclonal antibody No.82a (mab82) was verified by measuring its effect on whole-cell currents recorded from HEK cells transfected with the vEF1ap-5'UTR-TRPV6wt_CMVp-mCherry vector and treated with mouse monoclonal anti-TRPV6 antibody No.82a (mab82). Mouse monoclonal anti-TRPV6 antibody No.82a was applied in a series of increasing concentrations (1:5000, 1:2000, 1:1000 followed by 1:500, and 1:200 dilutions) to establish a dose-dependent effect of this antibody on TRPV6 currents. In Figure 8C, the observed TRPV6 currents with increasing concentrations of the applied antibody are summarized, suggesting a concentration-dependent effect.

[0224] Other monoclonal antibodies against epitope P2 directly affect TRPV6-induced currents Preincubation of cells with humanized mab82 or P2R4G08 resulted in a reduction in capacitative calcium influx above control, indicating that TRPV6-mediated calcium influx is reduced by humanized mab82 or P2R4G08, similar to mouse mab82 (Figure 14).

[0225] Monoclonal antibodies against epitope P3 directly affect TRPV6-induced currents Preincubation of cells with P3R4F03, P3R4E11 or P3R5H03 resulted in an increase in capacitative calcium influx above controls, indicating that TRPV6-mediated calcium influx is increased by P3R4F03, P3R4E11 or P3R5H03 (Figure 13).

[0226] Example 4 Treatment with anti-TRPV6 antibodies raised against extracellular epitopes reduces cell survival through modulation of TRPV6 activity antibody 79 Having established the direct effect of the antibody on the TRPV6 channel, the next question was whether the rabbit polyclonal anti-TRPV6 antibody No. 79a could affect PCa cell survival in vitro. For this, LNCaP cells were incubated with glycerol (CT) or with various dilutions of the polyclonal anti-TRPV6 antibody No. 79a or the control antibody anti-HA for 72 hours, and cell survival was measured by MTS assay (Figure 9A). A strong decrease in cell survival was observed with the polyclonal antibody 79a, whereas no effect was observed with the control anti-HA antibody.

[0227] As the cell viability assay based on the assessment of cytochrome p-450 activity is a complex assay, a panel of additional techniques was used to measure both cell proliferation and cell death.

[0228] Cell counting assays using various control antibodies (rabbit polyclonal anti-SERCA2B; rabbit polyclonal antibody No. 80 and rabbit polyclonal antibody No. 81 targeting an intracellular epitope of the TRPV6 channel; peptide 80 (hTRPV6 64-78; QRRESWAQSRDEQNL (SEQ ID NO: 189); peptide 81 hTRPV6 692-707; HTRGSEDLDKDSVEKL (SEQ ID NO: 190); rabbit polyclonal anti-GFP of the same isotype) confirm the results of the viability assay and highlight the specificity of polyclonal antibody No. 79a (Figure 9B).

[0229] The specificity of this effect due to anti-TRPV6 antibody No. 79 (79a) was demonstrated by cell viability assay (MTS) of LNCaP cells treated for 3 days with either anti-TRPV6 antibody No. 79 (79a) or a commercial anti-TRPV6 antibody (Alomone #ACC-036), with medium and an equivalent amount of glycerol as controls (CT) (Figure 9C).

[0230] Classical apoptosis assays by Hoechst staining were used to confirm the hypothesis of apoptosis induction by polyclonal anti-TRPV6 antibody No. 79a. 1 μM thapsigargin was used as a positive control for 3 days to induce calcium-dependent apoptosis by long-term treatment. Quantification of apoptotic cells showed a significant cell death rate induced by 3 days of treatment with polyclonal anti-TRPV6 antibody No. 79a for LNCaP cells (FIG. 10A) and HEK cells (FIG. 10B). The HEK cells are much more susceptible to apoptosis compared to PCa cells. In addition, the presence of TG simultaneously with polyclonal antibody No. 79a significantly enhanced apoptosis compared to treatment with TG alone in LNCaP for antibody 79 (FIG. 10A) and HEK for antibody 79a (FIG. 10B).

[0231] Cell cycle assays showed a clear subG1 peak in LNCaP cells treated with polyclonal antibody No. 79a, suggesting that anti-TRPV6 antibodies act through induction of apoptosis rather than reduction of proliferation (Figure S10C). Finally, to exclude necrosis as a possible mechanism, trypan blue staining was performed over an 8-, 24-, and 48-h time series, with stained cells appearing late, indicative of mid-to-late apoptosis with compromised membrane integrity (Figure S10D).

[0232] Antibody 82 (Ab82) Once the direct effect of the antibody on the TRPV6 channel was established, the next question was whether the mouse monoclonal anti-TRPV6 antibody mab82 could affect cancer cell survival in vitro. For this, LNCaP cells were incubated with glycerol (CT) or either the anti-TRPV6 antibody mab82 or the control antibody mabAU1 for 24, 48, 72, and 96 hours, and cell survival was measured by CellTiter (Figure 11). At 72 and 96 hours, a strong decrease in cell survival was observed with Mab82, but no effect was observed with the control mabAU1.

[0233] Monoclonal antibody against epitope P3 Once the antibody's cellular calcium modulation was established, the next question was whether the anti-TRPV6 antibody P3R4F03 could affect PCa cell survival in vitro. For this, LNCaP cells were incubated with either P3R4F03 antibody or an irrelevant antibody (IA) at various dilutions for 72 hours, and cell survival was measured by Cell titer glo assay (Figure 15). A decrease in cell survival was observed with P3R4F03 antibody compared to the irrelevant antibody (IA).

[0234] Example 5 Treatment with anti-TRPV6 antibodies raised against an extracellular epitope inhibits tumor growth and metastatic progression The therapeutic effect of monoclonal antibody 82 (mab82) was evaluated in PCa cells using PC3M trpv6- / - -pmCherry cell line and PC3M trp- / - The antibody treatment was performed using an immunodeficient "Swiss nude" mouse model implanted with 2x10E6 cells derived from a stable clone of the -pTRPV6wt cell line (Raphael et al., 2014). Antibody treatment was performed using two groups of PC3M cells with and without TRPV6 channels, and two subgroups in each group for treatment with either the control mouse monoclonal anti-AU1 antibody or the anti-TRPV6 antibody mab82. To facilitate in vivo monitoring, the antibodies were pre-conjugated to the Cf790 fluorophore. Kinetic studies were performed with different doses (0.5μg-15μg / mouse) determined based on the closest published literature (Bleeker et al., Br J Haematol., 2008, 140, 303-12). EC 50 (minimal dose required to maintain maximum duration) was calculated and a dose of 100 μg / kg (3 μg per mouse) was selected for both mab82 and the control antibody anti-AU1 of the same isotype (IgG2a). We also examined the biodistribution of both antibodies immediately after bolus injection and their distribution in different organs of the body 30 minutes after antibody injection. As soon as tumors were visualized, treatment with either the control anti-AU1 or the mab82 experimental antibody was started twice a week and gave the following convincing data: PC3M trpv6- / - Tumors in the -pTRPV6wt subgroup treated with mab82 were reduced in size and left only a barely visible blue border or none at 3 weeks after treatment initiation compared to the control anti-AU1 treated subgroup. After 5 weeks of treatment with mab82, this blue border had disappeared, leaving some connective tissue-like traces or none at all. Overall, with treatment starting on day 9 (arrows, FIG. 12A), significant differences in size were already seen starting on day 24 post-implantation and day 15 post-treatment, and at the end of the experiment, this difference was striking. PC3M trp- / -Tumors in the anti-AU1-treated subgroup of the -pTRPV6wt group were highly aggressive (due to the TRPV6 channel), reflecting the general survival rate (Figure 12B), whereas PC3M trpv6- / - It should be noted that the -pmCherry group did not show any significant differences between subgroups, regardless of the subgroup (antibody treatment) (Figure 12C). trpv6- / - -pmCherry group and PC3M trpv6- / - Regarding the difference in tumor growth between the -pTRPV6wt group, the latter group showed a significant difference in tumor growth due to the presence of TRPV6 channels (Figure 12D). In vivo mCherry imaging was performed weekly using a small animal imaging system to demonstrate tumor suppression. When tumors reached their maximum size, they were immediately excised, and in vivo imaging was continued weekly for at least 3 months while mice were kept alive.

[0235] Transfer test showed that PC3M trpv6- / - There were no metastases in the -pmCherry group, whereas PC3M treated with the control AU1 antibody trpv6- / - - 100% in the pTRPV6wt group, and 100% in the mab82-treated PC3M trp- / - It was clearly shown that there was only a 40% metastatic potential in the -pTRPV6wt group (Figure 2E). In this group, 70% of metastases were due to the presence of high-grade tumors in the neck, which are usually hypervascularized and difficult to resect due to excessive proliferation.

[0236] As a result, mab82 treatment has been demonstrated to be a promising therapeutic solution against solid tumor growth and the development of metastases in vivo.

[0237] A brief description of additional amino acid sequences useful in the practice of the present invention SEQ ID NO: 1: Human TRPV6 protein (UniProtKB / Swiss-Prot: NP_061116.5 or Q9H1D0.3)

[0238] [ka]

[0239]

Table 4A

[0240]

Table 4B

[0241]

Table 4C

[0242]

Table 4D

[0243]

Table 4E

[0244] [Table 4F]

[0245] Table 4G

[0246]

Table 4H

[0247]

Table 4I

[0248]

Table 4J

[0249]

Table 4K

[0250]

Table 4L

[0251]

Table 4M

[0252]

Table 4N

Entrusted number

[0253] UniProtKB / Swiss-Prot NP_061116.5 UniProtKB / Swiss-Prot Q9H1D0.3

Claims

**Claim 1**: An isolated antibody against human TRPV6 channel protein, comprising a heavy chain variable CDR containing VH-CDR1 of SEQ ID NO: 158, VH-CDR2 of SEQ ID NO: 159, and VH-CDR3 of SEQ ID NO: 160, and a light chain variable CDR containing VL-CDR1 of SEQ ID NO: 148, VL-CDR2 with the amino acid sequence GDS, and VL-CDR3 of SEQ ID NO:

149. **Claim 2**: The isolated antibody according to claim 1, which binds to the extracellular epitope of the hTRPV6 protein of SEQ ID NO:

1. **Claim 3**: The isolated antibody according to claim 1 or 2, which binds to an epitope derived from the third extracellular region of human TRPV6 of SEQ ID NO:

16. **Claim 4**: The isolated antibody according to claim 1, which regulates the activity of the human TRPV6 channel. **Claim 5**: The isolated antibody according to claim 1, which inhibits the proliferation of TRPV6-expressing cancer cells. **Claim 6**: The antibody according to claim 1, which is a humanized monoclonal antibody comprising a heavy chain variable domain containing VH-FR1 of SEQ ID NO: 161, VH-FR2 of SEQ ID NO: 162, VH-FR3 of SEQ ID NO: 163, and VH-FR4 of SEQ ID NO: 164, and a light chain variable domain containing VL-FR1 of SEQ ID NO: 150, VL-FR2 of SEQ ID NO: 151, VL-FR3 of SEQ ID NO: 152, and VL-FR4 of SEQ ID NO: 153 or 154. **Claim 7**: The isolated antibody according to claim 1, comprising a heavy chain variable domain sequence and a light chain variable domain sequence that each have at least 90% identity with the sequence pair: SEQ ID NO: 165 and SEQ ID NO: 155 or 156, with respect to the heavy chain variable domain sequence and the light chain variable domain sequence. **Claim 8**: The isolated antibody according to claim 7, comprising a heavy chain variable domain sequence and a light chain variable domain sequence that each have the sequence pair: SEQ ID NO: 165 and SEQ ID NO: 155 or 156. **Claim 9**: The isolated antibody according to claim 7, comprising a heavy chain sequence and a light chain sequence that each have at least 90% identity with the sequence pair: SEQ ID NO: 166 and SEQ ID NO: 157, with respect to the heavy chain sequence and the light chain sequence. **Claim 10**: The isolated antibody according to claim 9, comprising a heavy chain sequence and a light chain sequence that each have the sequence pair: SEQ ID NO: 166 and SEQ ID NO:

157. **Claim 11**: The isolated antibody according to claim 1, which is a humanized monoclonal antibody of human IgG1 or IgG4 isotype. **Claim 12**: The isolated antibody according to claim 1, which is conjugated with a labeling agent or a therapeutic agent. **Claim 13**: An expression vector for the recombinant production of the antibody in a host cell, comprising at least one nucleic acid encoding the heavy chain and / or light chain of the antibody according to claim 1.

14. A pharmaceutical composition comprising at least the antibody according to claim 10 and a pharmaceutically acceptable solvent.

15. A pharmaceutical composition comprising the antibody according to claim 1.

16. A pharmaceutical composition for the therapeutic treatment of cancer associated with TRPV6 expression, comprising the antibody according to claim 1.

17. A pharmaceutical composition for the in vitro diagnosis or prognosis prediction of cancer associated with TRPV6 expression, comprising the antibody according to claim 1.

18. The pharmaceutical composition for use according to claim 16 or 17, wherein the cancer is selected from the group consisting of endometrial cancer, leukemia, and cancers of the breast, pancreas, prostate, colon, ovary, and thyroid.

19. The pharmaceutical composition for use according to claim 18, wherein the cancer is prostate cancer.

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